Flavonoid and anthocyanin bioproduction using microorganism hosts

ABSTRACT

The invention is directed to methods involved in the production of flavonoids, anthocyanins and other organic compounds. The invention provides cells engineered for the production of flavonoids, anthocyanins and other organic compounds, where the engineered cells include one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts.

I. RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.63/174,403, filed on Apr. 13, 2021. The content of U.S. ProvisionalApplication No. 63/174,403 is hereby incorporated by reference in itsentirety.

II. SEQUENCE LISTING

This application contains a sequence listing filed in electronic form asan ASCII.txt file entitled DEBU-009-05-US-Sequence-Listing.txt, createdon Mar. 21, 2022, last modified Apr. 13, 2022, and having a size of 448KB. The content of the sequence listing is incorporated herein itsentirety.

III. FIELD OF THE INVENTION

The invention related to materials (including engineered cells and celllines) and methods involved in the production of flavonoids,anthocyanins and other organic compounds.

IV. BACKGROUND OF THE INVENTION

Flavonoids and anthocyanins are natural products produced in plants thatfind a variety of roles such as antioxidants, ultraviolet (UV) defensemechanisms, and colors. Over the past several years, the health benefitsof flavonoids and anthocyanins have been widely demonstrated. Thesecompounds are capable of scavenging radicals and can act as enzymeinhibitors and anti-inflammatory agents. With these recognized healthand color benefits, much research has gone into understanding how thesecompounds are made in nature. Flavonoids and anthocyanins aresynthesized from phenylpropanoid starter units and malonyl-Cofactor-A(malonyl-CoA) extender units that then undergo modifications to createmany polyphenol compounds such as taxifolin, naringenin, and(+)-catechin. However, in most cases, these compounds are extracted orchemically manufactured.

V. SUMMARY OF THE INVENTION

To move away from agriculture and chemically derived products, we havecreated engineered cells for the bioproduction of flavonoids andanthocyanins. This approach provides a feasible route for the rapid,safe, economical, and sustainable production of a wide variety ofimportant flavonoids.

Herein, a range of flavonoids and anthocyanins including naringenin,eriodictyol, taxifolin, dihydrokaempferol, (+)-catechin, cyanidin, andcyaninidin-3-glucoside are biomanufactured using a modified microbialhost. Herein, the engineered cells include one or more geneticmodifications that increase(s) flavonoid and anthocyanin bioproductionby increasing metabolic flux to flavonoid precursors and/or reducingcarbon losses resulting from the production of byproducts.

Provided herein are cells engineered for the production of flavonoids,anthocyanins and other organic compounds, where the engineered cellsinclude one or more genetic modifications that increase flavonoidproduction by increasing metabolic flux to flavonoid precursors and/orreducing carbon losses resulting from the production of byproducts. Asnonlimiting examples, a genetic modification can be a modification forover-expressing or under-expressing one or more endogenous genes in theengineered host cell or can be a modification for expressing one or morenon-native genes in the engineered host cell. Engineered cells asprovided herein can include multiple genetic modifications.

Also provided are cell cultures for producing one or more flavonoids oranthocyanins. The cell cultures include engineered cells as disclosedherein in a culture medium that includes a carbon source that can alsobe an energy source, such as glycerol, sugar, or an organic acid. Invarious embodiments, the culture medium can include at least one feedmolecule such as but not limited to one or more organic acids or aminoacids that can be converted into a flavonoid precursor (such astyrosine, p-coumaroyl-CoA or malonyl-CoA). Examples of feed moleculesinclude, but are not limited to, acetate, malonate, tyrosine,phenylalanine, pantothenate, coumarate, etc. In some embodiments, thefeed molecules may be of reduced or low purity. For example, glycerol asa feed molecule may be crude glycerol, including a biomass comprisingglycerol, for example, glycerol obtained as a byproduct of biodieselprocessing. Alternatively, or in addition, the culture medium caninclude a supplemental compound that can be a cofactor or a precursor ofa cofactor used by an enzyme that functions in a flavonoid pathway, suchas, for examples, bicarbonate, biotin, thiamine, pantothenate,alpha-ketoglutarate, ascorbate, or 5-aminolevulinic acid.

Further provided are methods for producing flavonoids and anthocyaninsthat include culturing a cell engineered for the production offlavonoids or anthocyanins as provided herein under conditions in whichthe cell produces flavonoids or anthocyanins. In some examples, themethods include culturing the engineered cells in a culture medium thatincludes at least one feed molecule or supplement such as but notlimited to: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate,acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate,and 5-aminolevulinic acid. The methods can further include recovering atleast one of the flavonoids from culture medium, whole culture, or thecells.

In a first aspect, provided herein are cells engineered to produce oneor more flavonoids or anthocyanins, wherein the cells include, inaddition to nucleic acid sequences encoding either tyrosine ammonialyase activity and/or phenylalanine ammonia lyase activity andcinnamate-4-hydroxylase activity, 4-coumarate-CoA ligase activity,chalcone synthase activity, chalcone isomerase activity,flavanone-3-hydroxylase activity, flavonoid 3′-hydroxylase activity orflavonoid 3′5′-hydroxylase activity, cytochrome P450 reductase activity,leucoanthocyanidin reductase activity, and dihydroflavonol-4-reductaseactivity, one or more genetic modifications for improving production ofthe flavonoids or anthocyanins. As set forth herein, a cell that isengineered to produce one or more of the flavonoids is engineered toinclude an exogenous nucleic acid sequence encoding tyrosine ammonialyase activity that can form 4-coumaric acid using tyrosine as substrate(e.g., tyrosine ammonia lyase TAL, EC: 4.3.1.25) or, alternatively or inaddition, an exogenous nucleic acid sequence encoding phenylalanineammonia lyase activity that can convert phenylalanine to trans-cinnamicacid and an exogenous nucleic acid sequence encodingcinnamate-4-hydroxylase activity that forms 4-coumaric acid fromtrans-cinnamic acid, an exogenous nucleic acid sequence encoding CoAligase activity that forms p-coumaroyl-CoA from coumaric acid (e.g.,4-coumarate-CoA ligase, 4CL, EC:6.2.1.12), an exogenous nucleic acidsequence encoding polyketide synthase activity that forms naringeninchalcone using malonyl-CoA and p-coumaroyl-CoA as substrates (e.g.,chalcone synthase, CHS, EC:2.3.1.74), an exogenous nucleic acid sequenceencoding chalcone isomerase activity that forms naringenin fromnaringenin chalcone via its cyclase activity (e.g., chalcone-flavononeisomerase, CHI, EC:5.5.1.6), an exogenous nucleic acid sequence encodingflavanone-3-hydroxylase activity that forms dihydrokaempferol fromnaringenin or forms taxifolin from eriodictyol (e.g., naringenin3-dioxygenase, F3H, EC: 1.14.11.9), an exogenous nucleic acid sequenceencoding flavonoid 3′-hydroxylase or flavonoid 3′5′-hydroxylase activitycoupled with an exogenous nucleic acid sequence encoding cytochrome P450reductase activity to form taxifolin or dihydromyricetin fromdihydrokaempferol or to form eriodictyol or pentahydroxyflavone fromnaringenin (e.g., flavonoid 3′-monooxygenase, F3′H, EC: 1.14.13.21, EC:1.14.14.82; cytochrome P450/NADPH-P450 reductase, EC:1.14.14.1; F3′5′H,EC:1.14.14.81), an exogenous nucleic acid sequence encodingdihydroflavonol-4-reductase activity that forms leucocyanidin fromtaxifolin, leucodelphinidin from dihydromyricetin, or leucopelargonidinfrom dihydrokaempferol (e.g., dihydroflavonol 4-reductase, EC:1.1.1),and an exogenous nucleic acid sequence encoding leucoanthocyanidinreductase activity that forms catechin from leucocyanidin (e.g.,leucoanthocyanidin reductase, LAR, EC:1.17.1.3). Optionally, a cell thatis engineered to produce anthocyanins is further engineered to includean exogenous nucleic acid sequence encoding anthocyanin synthaseactivity that forms cyanidin from catechin or leucocyanidin, formsdelphinidin from leucodelphinidin, or forms pelargonidin fromleucopelargonidin (e.g., anthocyanin synthase, ANS, EC:1.14.20.4) and toinclude an exogenous nucleic acid sequence encoding glucosyltransferaseactivity that forms cyanidin-3-O-beta-D-glucoside from cyanidin,delphinidin-3-O-beta-D-glucoside from delphinidin, orpelagonidin-3-O-beta-D-glucoside from pelagonidin (e.g., anthocyanidin3-O-glucosyltransferase, 3GT, EC:2.4.1.115). The cells provided hereinthat are engineered to produce flavonoids or anthocyanins are furtherengineered to increase the production of flavonoids or anthocyaninsproduct, for example by increasing metabolic flux to a flavonoid oranthocyanin pathway, or by decreasing byproduct formation.

A cell engineered to produce a flavonoid is further engineered toincrease the supply of precursor malonyl-CoA. One strategy forincreasing malonyl-CoA includes increasing acetyl-CoA carboxylase (ACC)activity. In various embodiments, the ACC enzyme, which in mosteukaryotes, including fungi, is a large single chain polypeptide, and inplant and bacteria such as E. coli is a multi-subunit enzyme, isoverexpressed in the host strain. Examples of acetyl-CoA carboxylasethat can be expressed in a host cell engineered to produce a flavonoidor anthocyanin include, without limitation, the ACC genes of Mucorcircinelloides, Rhodotorula toruloides, Lipomyces starkeyi, Ustilagomaydis, and orthologs of these ACCs in other species having at least 50%amino acid identity to these ACCs.

Additional strategies for increasing malonyl-CoA include increasingacetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase(ACC). In some embodiments, acetyl-CoA synthase (ACS) that convertsacetate and CoA to acetyl-CoA is over-expressed in the host cells.Cultures of engineered host cells that include overexpressed nucleicacid sequence encoding ACS can optionally include acetate in the culturemedium. Examples of acetyl-CoA synthase that can be expressed in a hostcell engineered to produce a flavonoid or anthocyanin include, withoutlimitation, the ACS gene of E. coli, the ACS of Salmonella typhimurium,orthologs of these ACSs in other species having at least 50% amino acididentity to these ACSs.

Also considered, in further embodiments, is an engineered host cell thatoverexpresses a gene encoding pyruvate dehydrogenase (PDH), whichconverts pyruvate to acetyl-CoA. Further, in E. coli, a variant of theLpd subunit of PDH can be expressed that includes a mutation (E354K)that reduces inhibition of PDH by NADH.

Alternatively, or in addition to strategies for increasing ACC activityand strategies for increasing acetyl-CoA, strategies for increasingmalonyl-CoA by mechanisms that do not rely on the activity of an ACC canbe employed. In some embodiments, a cell engineered to produce aflavonoid, or an anthocyanin, is further engineered to increase thecell's supply of malonyl-CoA includes an exogenous nucleic acid sequenceencoding a malonyl-CoA synthetase that generates malonyl-CoA frommalonate. Examples of malonyl-CoA synthetases include the malonyl-CoAsynthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or amalonyl-CoA synthetase having at least 50% identity to any of these orother naturally occurring malonyl-CoA synthetases. Malonate canoptionally be added to the culture medium of a culture that includes acell engineered to express a malonyl-CoA synthetase. An engineered cellthat includes an exogenous gene encoding a malonyl-CoA synthetase canalso include an exogenous nucleic acid sequence encoding a malonatetransporter, such as a malonate transporter encoded by a matC gene, forexample, of Streptomyces coelicolor, or a malonate transporter encodedby DctPQM of Sinorhizobium medicae.

In additional embodiments, a cell engineered to produce a flavonoid oran anthocyanin is further engineered to include an exogenous nucleicacid sequence encoding malonate CoA-transferase that makes malonyl-CoAby direct transfer of the CoA from acetyl-CoA. Examples of malonateCoA-transferase that can be expressed in an engineered cell as providedherein include, without limitation, the alpha subunit (mdcA) of malonatedecarboxylase from Acinetobacter calcoaceticus, Geobacillus sp, or atransferase having at least 50% identity to any of these or othernaturally occurring malonate CoA-transferases.

In some embodiments, a cell engineered to produce flavonoids oranthocyanins is further engineered to increase the supply of coenzyme A(CoA) to increase its availability for producing acetyl-CoA,malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoAsupply include upregulating endogenous pantothenate kinase (PanK)(EC:2.7.1.33) that produces CoA from pantothenate. Alternatively, or inaddition, a host cell can be engineered to include a nucleic acidsequence encoding type III pantothenate kinase that is not feedbackinhibited by coenzyme A, such as CoaX gene of Pseudomonas aeruginosa(EC:2.7.1.33). Cultures of cells engineered for the production offlavonoids or anthocyanins can in some embodiments include a medium thatincludes pantothenate, a precursor of CoA biosynthesis, and canoptionally also include cysteine, used in the CoA biosynthesis.

Additional strategies to increase malonyl-CoA flux to the flavonoidpathway include mutation or downregulation of one or more genes thatfunction in fatty acid biosynthesis. Without limiting the embodiments toany particular mechanism, limiting fatty acid biosynthesis can increasethe malonyl-CoA supply available for flavonoid biosynthesis. In someembodiments, the gene beta-ketoacyl-ACP synthase II (E. coli fabF) canbe disrupted to reduce fatty acid biosynthesis. Another example of afatty acid biosynthesis gene of a host cell that may be mutated ordownregulated is a gene encoding malonyl-CoA-ACP transacylase (E. colifabD). Other fatty acid biosynthesis genes of the engineered host cellthat can be downregulated include a beta-ketoacyl-ACP synthase I enzyme(E. coli fabB) and acyl carrier protein (E. coli acpP).

Additional genetic modifications that may be present in a host cellengineered to produce flavonoids or anthocyanins include downregulation,disruption, or deletion of genes encoding alcohol dehydrogenase, lactatedehydrogenase, pyruvate oxidase, acetyl phosphate transferase andacetate kinase. In an E. coli host cell, genes that are downregulated,disrupted, or deleted can include aldehyde-alcohol dehydrogenase (adhE),lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), and enzymeacetate kinase phosphate acetyltransferase (ackA-pta).

Further, a cell engineered for the production of flavonoids oranthocyanins can have one or more genes encoding thioesterasesdownregulated, disrupted, or deleted to prevent hydrolysis of precursorsmalonyl-CoA, actetyl-CoA, and/or p-coumaryol-CoA. For example, in an E.coli host one or more of the thioesterase genes tesA, tesB, yciA, andybgC can be downregulated, disrupted, or deleted.

Alternatively, or in addition, genes encoding enzymes of thetricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can bedisrupted or downregulated to increase alpha-ketoglutarate supply whichserves as a cofactor for one or more of the flavonoid and anthocyaninpathway enzymes. Other TCA enzymes that can be downregulated includecitrate synthase that converts acetyl-CoA to citrate.

Also considered, in further embodiments, is an engineered host cell forthe production of flavonoids or anthocyanins to upregulate theendogenous biosynthesis of amino acid tyrosine. Tyrosine is one of theprecursors for the flavonoid biosynthesis and its conversion to coumaricacid is the first committed step of the pathway. L-tyrosine is one ofthe three aromatic amino acids derived from the shikimate pathway. Theinitial step of the shikimate pathway is catalyzed by DAHP synthaseisozymes and regulated through feedback-inhibition. Strategies toincrease tyrosine production can include, without limitation,transcriptional deregulation, removing feedback inhibition,overexpression of rate-limiting enzymes, and/or deletion of theL-phenylalanine branch of the aromatic acid biosynthetic pathway. Forexample, in an E. coli host the tyrR gene can be disrupted,feedback-inhibition-resistant versions of the DAHP synthase (aroG) andchorismate mutase (tyrA) can be introduced, and/or rate-limitingenzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimatedehydrogenase (ydiB) can be overexpressed. Further, thePhosphoenolpyruvate synthase (ppsA) and transketolase (tktA) can beexogenously introduced to enhance tyrosine production.

Also considered, in further embodiments, is an engineered host cell forthe production of flavonoids or anthocyanins further engineered toupregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450(CYPs), one of the exogenous genes in the engineered cells providedherein, contain heme as a cofactor. Improving heme supply can be aneffective strategy to increase flavonoid biosynthesis. 5-aminolevulinicacid (ALA) is the first committed precursor to the heme pathway.Strategies to increase heme supply include overexpression of the genesthat synthesize the precursor ALA. In an E. coli host, ALA is formedfrom the 5-carbon skeleton of glutamate (the C5 pathway). The threeenzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase(gltX), glutamyl-tRNA reductase (hemA), and glutamate-1-semialdehydeaminotransferase (hemL). In an E. coli host, the engineered cellsprovided herein can be further engineered to express or overexpress hemAor its variants, and/or hemL to increase the heme precursor ALAproduction. The nonlimiting examples of hemA gene that can beoverexpressed include a mutated hemA (inserting two lysine residualsbetween Thr-2 and Leu-3 at N terminus of hemA gene from Salmonellatyphimurium (EC:1.1.1.70). Alternatively, or in addition, a heterologousALAS gene can be introduced to produce ALA via the C4 pathway (ALS issynthesized by the condensation of glycine and succinyl-CoA).Nonlimiting examples of heterologous ALAS that can be expressed in E.coli include ALAS of Bradyrhizobium japonicum (EC: 2.3.1.37), ALAS ofRhodobacter capsulatus, or an ALAS having at least 50% sequence identityto a naturally occurring ALAS. Further, one or more of the downstreamgenes (e.g., in E. coli hemB, hemC, hemD, hemE, hemF, hemG, hemI, orhemH) that catalyze the synthesis of heme from ALA can be overexpressedto drive the flux from ALA to heme production. Cultures of cellsengineered for the production of flavonoids or anthocyanins can in someembodiments include a medium that includes succinate and/or glycine,precursors of heme biosynthesis via the C4 pathway.

In another aspect, provided herein are cell cultures that includeengineered cells as provided herein in a culture medium, where theculture medium includes a carbon source that is also an energy sourcefor the cells, where the carbon source can be, for example, glycerol, asugar, or an organic acid, as nonlimiting examples. The culture mediumcan further include a feed molecule that is used to produce flavonoidsor anthocyanins. A feed molecule can be, for example, acetate, malonate,tyrosine, pantothenate, coumarate, biotin, alpha-ketoglutarate,ascorbate, 5-aminolevulinic acid, succinate, or glycine. In someembodiments, the culture comprises a culture medium that includes acarbon source and at least one supplement that is a cofactor of anenzyme or is a precursor of an enzyme cofactor.

In yet another aspect, methods for producing flavonoids and anthocyaninsthat include incubating a culture of engineered host cell as providedherein to produce flavonoids or anthocyanins. The methods can furtherinclude recovering at least one of the flavonoids from the cells, theculture medium, or the whole culture.

In yet another aspect, the invention provides an engineered host cellthat comprises one or more genetic modifications resulting in productionof flavonoid or anthocyanin from a carbon source that can also be anenergy source, through multiple chemical intermediates, by theengineered host cell. In certain embodiments, the production offlavonoid or anthocyanin from glycerol occurs through enzymatictransformation. In certain embodiments, the production of flavonoid oranthocyanin from a carbon source that can also be an energy sourceoccurs through enzymatic transformation. In certain embodiments, thecarbon source is selected from a group consisting of: (i) glycerol, (ii)a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomasscomprising glycerol; and (vi) any combination thereof. In certainembodiments, the engineered host cell is cultured in a medium comprisingmolecules selected from a group consisting of: (i) glycerol, (ii) asugar, (iii) an organic acid, (iv) an amino acid, (v) a biomasscomprising glycerol; and (vi) any combination thereof. In certainembodiments, one or more genetic modifications lead to increasemetabolic flux to flavonoid precursors or cofactors. In certainembodiments, one or more genetic modifications cause reduction offormation of byproducts. In certain embodiments, one or more geneticmodifications are selected from: (i) one or more modifications forover-expressing one or more endogenous genes in the engineered hostcells; (ii) one or more modifications for under-expressing one or moreendogenous genes in the engineered host cells; (iii) one or more geneticmodification is expressing one or more non-native genes in theengineered host cells; and (iv) a combination thereof. In certainembodiments, the engineered host cell is cultured in a medium comprisingmolecules selected from: tyrosine, phenylalanine, malonate, p-coumarate,bicarbonate, acetate, pantothenate, biotin, thiamine,alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein oneor more of the selected molecules are the chemical intermediates,including molecules in the biosynthesis pathway or cofactors. In certainembodiments, the engineered host cell comprises at least one or morenucleic acid sequences selected from: (i) nucleic acid sequencesencoding tyrosine ammonia lyase activity; (ii) nucleic acid sequencesencoding phenylalanine ammonia lyase activity; (iii) nucleic acidsequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acidsequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) anycombination thereof. In certain embodiments, the engineered host cellcomprises at least one or more peptides selected from: (i) chalconeisomerase; (ii) chalcone synthase; (iii) a fusion protein comprises achalcone synthase and a chalcone isomerase; and (iv) any combinationthereof. In certain embodiments, the engineered cell is E. coli. Incertain embodiments, one or more genetic modifications decreases fattyacid biosynthesis. In certain embodiments, the engineered host cellcomprises an exogenous nucleic acid sequence selected from: (i) nucleicacid sequence encoding tyrosine ammonia lyase, wherein the encodedtyrosine ammonia lyase forms 4-coumaric acid using tyrosine as asubstrate; (ii) nucleic acid sequence encoding phenylalanine ammonialyase, wherein the encoded phenylalanine ammonia lyase convertsphenylalanine to trans-cinnamic acid; (iii) nucleic acid sequenceencoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylaseproduces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acidsequence encoding flavanone-3-hydroxylase, whereinflavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v)any combinations thereof. In certain embodiments, the engineered hostcell comprises an exogenous nucleic acid sequence selected from thegroup consisting of: (i) nucleic acid sequences encoding tyrosineammonia lyase, wherein the encoded tyrosine ammonia lyase forms4-coumaric acid using tyrosine as a substrate; (ii) nucleic acidsequence encoding phenylalanine ammonia lyase, wherein the encodedphenylalanine ammonia lyase converts phenylalanine to trans-cinnamicacid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase,wherein the cinnamate-4-hydroxylase produces 4-coumaric acid fromtrans-cinnamic acid; (iv) nucleic acid sequence encoding4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase formsp-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encodingchalcone synthase activity, wherein chalcone synthase forms naringeninchalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acidsequence encoding chalcone isomerase activity, wherein chalconeisomerase forms naringenin from naringenin chalcone; (vii) nucleic acidsequence encoding flavanone-3-hydroxylase, whereinflavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and(viii) any combinations thereof. In certain embodiments, the flavonoidis catechin.

In yet another aspect, the invention provides a method of increasing theproduction of flavonoids or anthocyanins, the method comprising:providing an engineered host cell that comprises one or more geneticmodifications resulting in production of flavonoid or anthocyanin from acarbon source that can also be an energy source, through multiplechemical intermediates, by the engineered host cell. In certainembodiments, the production of flavonoid or anthocyanin from a carbonsource that can also be an energy source occurs through enzymatictransformation. In certain embodiments, the carbon source is selectedfrom a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organicacid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi)any combination thereof. In certain embodiments, the engineered hostcell is cultured in a medium comprising molecules selected from a groupconsisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv)an amino acid, (v) a biomass comprising glycerol; and (vi) anycombination thereof. In certain embodiments, one or more geneticmodifications lead to increase metabolic flux to flavonoid precursors orcofactors. In certain embodiments, one or more genetic modificationscause increased metabolic flux to flavonoid precursors. In certainembodiments, one or more genetic modifications cause reduction in theformation of byproducts. In certain embodiments, one or more geneticmodifications are selected from: (i) one or more modifications forover-expressing one or more endogenous genes in the engineered hostcells; (ii) one or more modifications for under-expressing one or moreendogenous genes in the engineered host cells; (iii) one or more geneticmodification is expressing one or more non-native genes in theengineered host cells; and (iv) a combination thereof. In certainembodiments, the engineered host cell is cultured in a medium comprisingmolecules selected from: tyrosine, phenylalanine, malonate, p-coumarate,bicarbonate, acetate, pantothenate, biotin, thiamine,alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein oneor more of the selected molecules are the chemical intermediates,including molecules in the biosynthesis pathway or cofactors. In certainembodiments, the engineered host cell comprises at least one or morenucleic acid sequences selected from: (i) a nucleic acid sequencesencoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequencesencoding phenylalanine ammonia lyase activity; (iii) cinnamate4-hydroxylase; and (iv) any combination thereof. In certain embodiments,the engineered host cell comprises at least one or more peptidesselected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) afusion protein comprises a chalcone synthase and a chalcone isomerase;and (iv) any combination thereof. In certain embodiments, the engineeredcell is E. Coli. In certain embodiments, one or more geneticmodifications decreases fatty acid biosynthesis. In certain embodiments,the engineered host cell comprises an exogenous nucleic acid sequenceselected from: (i) nucleic acid sequences encoding tyrosine ammonialyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acidusing tyrosine as a substrate; (ii) nucleic acid sequence encodingphenylalanine ammonia lyase, wherein the encoded phenylalanine ammonialyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acidsequence encoding cinnamate-4-hydroxylase, wherein thecinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamicacid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase,wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin;and (v) any combinations thereof. In certain embodiments, the engineeredhost cell comprises an exogenous nucleic acid sequence selected from thegroup consisting of: (i) nucleic acid sequence encoding tyrosine ammonialyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acidusing tyrosine as a substrate; (ii) nucleic acid sequence encodingphenylalanine ammonia lyase, wherein the encoded phenylalanine ammonialyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acidsequence encoding cinnamate-4-hydroxylase, wherein thecinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamicacid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligaseactivity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA fromcoumaric acid (v) nucleic acid sequence encoding chalcone synthaseactivity, wherein chalcone synthase forms naringenin chalcone frommalonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encodingchalcone isomerase activity, wherein chalcone isomerase forms naringeninfrom naringenin chalcone; (vii) nucleic acid sequence encodingflavanone-3-hydroxylase, wherein flavanone-3-hydroxylase formsdihydrokaempferol from naringenin; and (viii) any combinations thereof.In certain embodiments, the flavonoid is catechin.

In yet another aspect, the invention provides a plurality of engineeredhost cells, wherein each of the plurality of the engineered host cellscomprises one or more genetic modifications resulting in production offlavonoid or anthocyanin from a carbon source that can also be an energysource, through multiple chemical intermediates. In certain embodiments,the production of flavonoid or anthocyanin from a carbon source that canalso be an energy source occurs through enzymatic transformation. Incertain embodiments, the carbon source is selected from a groupconsisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv)an amino acid, (v) a biomass comprising glycerol; and (vi) anycombination thereof. In certain embodiments, the engineered host cell iscultured in a medium comprising molecules selected from a groupconsisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv)an amino acid, (v) a biomass comprising glycerol; and (vi) anycombination thereof. In certain embodiments, one or more geneticmodifications lead to increase metabolic flux to flavonoid precursors orcofactors. In certain embodiments, one or more genetic modificationslead to increase metabolic flux to flavonoid precursors or cofactors. Incertain embodiments, one or more genetic modifications cause reductionof formation of byproducts. In certain embodiments, one or more geneticmodifications are selected from: (i) one or more modifications forover-expressing one or more endogenous genes in the engineered hostcells; (ii) one or more modifications for under-expressing one or moreendogenous genes in the engineered host cells; (iii) one or more geneticmodification is expressing one or more non-native genes in theengineered host cells; and (iv) a combination thereof. In certainembodiments, at least one of the engineered cells from the plurality ofthe engineered host cells is cultured in a medium comprising moleculesselected from: tyrosine, phenylalanine, malonate, p-coumarate,bicarbonate, acetate, pantothenate, biotin, thiamine,alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein oneor more of the selected molecules are the chemical intermediates,including molecules in biosynthesis pathway or cofactors. In certainembodiments, at least one of the engineered cells from the plurality ofthe engineered host cells comprise at least one or more nucleic acidsequences selected from: (i) nucleic acid sequences encoding tyrosineammonia lyase activity; (ii) nucleic acid sequences encodingphenylalanine ammonia lyase activity; (iii) nucleic acid sequencesencoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequencesencoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combinationthereof. In certain embodiments, at least one of the engineered hostcell from the plurality of engineered host cells comprise at least oneor more peptides selected from: (i) chalcone isomerase; (ii) chalconesynthase; (iii) a fusion protein comprises a chalcone synthase and achalcone isomerase; and (iv) any combination thereof. In certainembodiments, at least one the engineered host cell is E. coli. Incertain embodiments, one or more genetic modifications decreases fattyacid biosynthesis. In certain embodiments, at least one of theengineered host cell from the plurality of the engineered host cellscomprises an exogenous nucleic acid sequence selected from: (i) nucleicacid sequence encoding tyrosine ammonia lyase, wherein the encodedtyrosine ammonia lyase forms 4-coumaric acid using tyrosine as asubstrate; (ii) nucleic acid sequence encoding phenylalanine ammonialyase, wherein the encoded phenylalanine ammonia lyase convertsphenylalanine to trans-cinnamic acid; (iii) nucleic acid sequenceencoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylaseproduces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acidsequence encoding flavanone-3-hydroxylase, whereinflavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v)any combinations thereof. In certain embodiments, the engineered hostcell comprises an exogenous nucleic acid sequence selected from thegroup consisting of: (i) nucleic acid sequence encoding tyrosine ammonialyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acidusing tyrosine as a substrate; (ii) nucleic acid sequence encodingphenylalanine ammonia lyase, wherein the encoded phenylalanine ammonialyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acidsequence encoding cinnamate-4-hydroxylase, wherein thecinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamicacid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligaseactivity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA fromcoumaric acid (v) nucleic acid sequence encoding chalcone synthaseactivity, wherein chalcone synthase forms naringenin chalcone frommalonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encodingchalcone isomerase activity, wherein chalcone isomerase forms naringeninfrom naringenin chalcone; (vii) nucleic acid sequence encodingflavanone-3-hydroxylase, wherein flavanone-3-hydroxylase formsdihydrokaempferol from naringenin; and (viii) any combinations thereof.In certain embodiments, the flavonoid is catechin.

In yet another aspect, the invention provides a method of increasing theproduction of flavonoids or anthocyanins, the method comprising:providing a plurality of engineered host cells, wherein each of theplurality of the engineered host cell comprises one or more geneticmodifications resulting production of flavonoid or anthocyanin from acarbon source that can also be an energy source, through multiplechemical intermediates, by the engineered host cell. In certainembodiments, the production of flavonoid or anthocyanin from a carbonsource that can also be an energy source occurs through enzymatictransformation. In certain embodiments, the carbon source is selectedfrom a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organicacid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi)any combination thereof. In certain embodiments, the engineered hostcell is cultured in a medium comprising molecules selected from a groupconsisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv)an amino acid, (v) a biomass comprising glycerol; and (vi) anycombination thereof. In certain embodiments, one or more geneticmodifications lead to increase metabolic flux to flavonoid precursors orcofactors. In certain embodiments, one or more genetic modificationslead to increase metabolic flux to flavonoid precursors or cofactors. Incertain embodiments, one or more genetic modifications cause reductionof formation of byproducts. In certain embodiments, one or more geneticmodifications are selected from: (i) one or more modifications forover-expressing one or more endogenous genes in the engineered hostcells; (ii) one or more modifications for under-expressing one or moreendogenous genes in the engineered host cells; (iii) one or more geneticmodification is expressing one or more non-native genes in theengineered host cells; and (iv) a combination thereof. In certainembodiments, at least one of the engineered cells from the plurality ofthe engineered host cells is cultured in a medium comprising moleculesselected from: tyrosine, phenylalanine, malonate, p-coumarate,bicarbonate, acetate, pantothenate, biotin, thiamine,alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein oneor more of the selected molecules are the chemical intermediates,including molecules in biosynthesis pathway or cofactors. In certainembodiments, at least one of the engineered cells from the plurality ofthe engineered host cells comprise at least one or more nucleic acidsequences selected from: (i) nucleic acid sequences encoding tyrosineammonia lyase activity; (ii) nucleic acid sequences encodingphenylalanine ammonia lyase activity; (iii) nucleic acid sequencesencoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequencesencoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combinationthereof. In certain embodiments, at least one of the engineered hostcell from the plurality of engineered host cells comprise at least oneor more peptides selected from: (i) chalcone isomerase; (ii) chalconesynthase; (iii) a fusion protein comprises a chalcone synthase and achalcone isomerase; and (iv) any combination thereof. In certainembodiments, at least one the engineered host cell is E. coli. Incertain embodiments, one or more genetic modifications decreases fattyacid biosynthesis. In certain embodiments, at least one of theengineered host cell from the plurality of the engineered host cellscomprises an exogenous nucleic acid sequence selected from: (i) nucleicacid sequence encoding tyrosine ammonia lyase, wherein the encodedtyrosine ammonia lyase forms 4-coumaric acid using tyrosine as asubstrate; (ii) nucleic acid sequence encoding phenylalanine ammonialyase, wherein the encoded phenylalanine ammonia lyase convertsphenylalanine to trans-cinnamic acid; (iii) nucleic acid sequenceencoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylaseproduces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acidsequence encoding flavanone-3-hydroxylase, whereinflavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v)any combinations thereof. In certain embodiments, the engineered hostcell comprises an exogenous nucleic acid sequence selected from thegroup consisting of: (i) nucleic acid sequence encoding tyrosine ammonialyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acidusing tyrosine as a substrate; (ii) nucleic acid sequence encodingphenylalanine ammonia lyase, wherein the encoded phenylalanine ammonialyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acidsequence encoding cinnamate-4-hydroxylase, wherein thecinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamicacid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligaseactivity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA fromcoumaric acid (v) nucleic acid sequence encoding chalcone synthaseactivity, wherein chalcone synthase forms naringenin chalcone frommalonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encodingchalcone isomerase activity, wherein chalcone isomerase forms naringeninfrom naringenin chalcone; (vii) nucleic acid sequence encodingflavanone-3-hydroxylase, wherein flavanone-3-hydroxylase formsdihydrokaempferol from naringenin; and (viii) any combinations thereof.In certain embodiments, the flavonoid is catechin.

In yet another aspect, the engineered host cell comprises one or moregenetic modifications to increase the production and/or availability ofmalonyl-CoA. In certain embodiments, the production and/or availabilityof malonyl-CoA is increased by transformation of acetyl-CoA tomalonyl-CoA. In certain embodiments, the engineered host cell comprisesone or more genetic modifications selected from: (i) expression ofacetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoAcarboxylase. In another embodiment, the engineered host cell is an E.coli. In certain embodiments, the E. coli cell further comprises genesfrom fungi. In certain embodiments, the acetyl-CoA carboxylase is from:Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, andUstilago maydis, and orthologs of acetyl-CoA carboxylase having at least50% amino acid identity to the acetyl-CoA carboxylase of theseaforementioned species. In certain embodiments, one or more geneticmodification is deletion or attenuation of one or more fattybiosynthetic genes resulting in decrease in fatty acid biosynthesis. Incertain embodiments, one or more genetic modification is overexpressionof acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoAsynthase is selected from: acetyl-CoA synthase gene of E. coli,acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs ofacetyl-CoA synthase gene in any other species having at least 50% aminoacid identity to the acetyl-CoA synthase gene of E. coli and Salmonellatyphimurium. In certain embodiments, one or more genetic modification isselected from a group consisting of: (i) overexpression a gene encodingpyruvate dehydrogenase (PDH), wherein the PDH may include E354Kmutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoAsynthetase; (iii) upregulation of endogenous pantothenate kinase (PanK),wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenousnucleic acid sequence encoding a malonate transporter; and (v) anycombinations thereof. In certain embodiments, the malonyl-CoA synthetaseis selected from of malonyl-CoA synthetases of Streptomyces coelicolor,Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least50% identity to any of these or other naturally occurring malonyl-CoAsynthetases. In certain embodiments, one or more genetic modificationsto decrease fatty acid biosynthesis is selected from: (i) mutation ordownregulation of a gene encoding malonyl-CoA-ACP transacylase (E. colifabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E.coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme(E. coli fabB); (iv) downregulation of acyl carrier protein (E. coliacpP); and (v) any combinations thereof. In certain embodiments, theengineered host cell comprises peptides selected from: (i) acetyl-CoAcarboxylase (ACC) having an amino acid sequence at least 80% identicalto the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii)malonate CoA-transferase having an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO: 19; (iii)acetyl-CoA synthase (ACS) having an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO: 16; (iv)malonyl-CoA synthase having an amino acid sequence at least 80%identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonatetransporter having an amino acid sequence at least 80% identical to SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinasehaving an amino acid sequence at least 80% identical to SEQ ID NO: 88,SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.

In another aspect, the invention provides a method of increasing theproduction of flavonoids comprising an engineered host cell, wherein theone or more engineered host cells comprise one or more geneticmodifications to increase the production and/or availability ofmalonyl-CoA. In certain embodiments, the production and/or availabilityof malonyl-CoA is increased by transformation of acetyl-CoA tomalonyl-CoA. In certain embodiments, the engineered host cell comprisesone or more genetic modifications selected from: (i) expression ofacetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoAcarboxylase. In another embodiment, the engineered host cell is an E.coli. In certain embodiments, the E. coli cell further comprises genesfrom fungi. In certain embodiments, the acetyl-CoA carboxylase is from:Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, andUstilago maydis, and orthologs of acetyl-CoA carboxylase having at least50% amino acid identity to the acetyl-CoA carboxylase of theseaforementioned species. In certain embodiments, one or more geneticmodification is deletion or attenuation of one or more fattybiosynthetic genes resulting in decrease in fatty acid biosynthesis. Incertain embodiments, one or more genetic modification is overexpressionof acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoAsynthase is selected from: acetyl-CoA synthase gene of E. coli,acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs ofacetyl-CoA synthase gene in any other species having at least 50% aminoacid identity to the acetyl-CoA synthase gene of E. coli and Salmonellatyphimurium. In certain embodiments, one or more genetic modification isselected from a group consisting of: (i) overexpression a gene encodingpyruvate dehydrogenase (PDH), wherein the PDH may include E354Kmutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoAsynthetase; (iii) upregulation of endogenous pantothenate kinase (PanK),wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenousnucleic acid sequence encoding a malonate transporter; and (v) anycombinations thereof. In certain embodiments, the malonyl-CoA synthetaseis selected from of malonyl-CoA synthetases of Streptomyces coelicolor,Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least50% identity to any of these or other naturally occurring malonyl-CoAsynthetases. In certain embodiments, one or more genetic modificationsto decrease fatty acid biosynthesis is selected from: (i) mutation ordownregulation of a gene encoding malonyl-CoA-ACP transacylase (E. colifabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E.coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme(E. coli fabB); (iv) downregulation of acyl carrier protein (E. coliacpP); and (v) any combinations thereof. In certain embodiments, theengineered host cell comprises peptides selected from: (i) acetyl-CoAcarboxylase (ACC) having an amino acid sequence at least 80% identicalto the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii)malonate CoA-transferase having an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO: 19; (iii)acetyl-CoA synthase (ACS) having an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO: 16; (iv)malonyl-CoA synthase having an amino acid sequence at least 80%identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonatetransporter having an amino acid sequence at least 80% identical to SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinasehaving an amino acid sequence at least 80% identical to SEQ ID NO: 88,SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.

In another aspect, the invention provides an engineered host cell,wherein the engineered host cell comprises one or more geneticmodifications to increase endogenous biosynthesis of tyrosine. Incertain embodiments, one or more genetic modifications comprisesupregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certainembodiments, one or more genetic modifications are selected from: (i)upregulation of chorismate mutase; (ii) upregulation of prephenatedehydrogenase; (iii) overexpression of shikimate kinase; (iv)overexpression of shikimate dehydrogenase; and (v) any combinationsthereof. In certain embodiments, one or more genetic modificationscomprises downregulation of L-phenylalanine biosynthetic pathway. Incertain embodiments, one or more genetic modifications comprisesexpression of exogenous phosphoenolpyruvate synthase (ppsA). In certainembodiments, one or more genetic modifications comprises expression ofexogenous transketolase (tktA). In certain embodiments, wherein the oneor more genetic modifications comprises disruption of tyrR gene. Incertain embodiments, one or more genetic modifications are selected froma group consisting of: (i) expression or overexpression of (D146N)variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expressionor overexpression of variant of 3-dehydroquinate synthase (aroB); (iii)overexpression of transketolase tktA; (iv) deletion of shikimate kinase(aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354Vvariant of chorismate mutase (tyrA); (vi) and any combination thereof.

In another aspect, the invention provides a method of increasingendogenous biosynthesis of tyrosine comprising an engineered cell,wherein the engineered host cell comprises one or more geneticmodifications to increase endogenous biosynthesis of tyrosine. Incertain embodiments, one or more genetic modifications comprisesupregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certainembodiments, one or more genetic modifications are selected from: (i)upregulation of chorismate mutase; (ii) upregulation of prephenatedehydrogenase; (iii) overexpression of shikimate kinase; (iv)overexpression of shikimate dehydrogenase; and (v) any combinationsthereof. In certain embodiments, one or more genetic modificationscomprises downregulation of L-phenylalanine biosynthetic pathway. Incertain embodiments, one or more genetic modifications comprisesexpression of exogenous phosphoenolpyruvate synthase (ppsA). In certainembodiments, one or more genetic modifications comprises expression ofexogenous transketolase (tktA). In certain embodiments, wherein the oneor more genetic modifications comprises disruption of tyrR gene. Incertain embodiments, one or more genetic modifications are selected froma group consisting of: (i) expression or overexpression of (D146N)variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expressionor overexpression of variant of 3-dehydroquinate synthase (aroB); (iii)overexpression of transketolase tktA; (iv) deletion of shikimate kinase(aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354Vvariant of chorismate mutase (tyrA); (vi) and any combination thereof.

In another aspect, the invention provides an engineered host cell,wherein the engineered host cell comprises one or more geneticmodifications to increase transformation of leucocyanidin or catechin tocyanidin-3-glucoside (Cy3G). In certain embodiments, one or more geneticmodifications comprises overexpression of anthocyanin synthase. Incertain embodiments, the anthocyanin synthase is selected from: (i)anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) theanthocyanin synthase has an amino acid sequence at least 80% identicalto SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69;(iii) the anthocyanin synthase has an amino acid sequence at least 80%identical to SEQ. ID NO: 13; and (iv) any combinations thereof. Incertain embodiments, one or more engineered host cells comprisesflavonoid-3-glucosyl transferase (3GT). In certain embodiments,flavonoid-3-glucosyl transferase is selected from: (i)flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii)the flavonoid-3-glucosyl transferase has an amino acid sequence at least80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ.ID NO: 73; and (iii) any combinations thereof. In certain embodiments,one or more genetic modifications comprises overexpression ofanthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). Incertain embodiments, the one or more genetic modifications comprisesoverexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more geneticmodifications are selected from a group consisting of: (i) anthocyaninsynthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) acombination thereof.

In another aspect, the invention provides a method for increasing theproduction of flavonoids comprising an engineered host cell, wherein theengineered host cell comprises one or more genetic modifications toincrease transformation of leucocyanidin or catechin tocyanidin-3-glucoside (Cy3G). In certain embodiments, one or more geneticmodifications comprises overexpression of anthocyanin synthase. Incertain embodiments, the anthocyanin synthase is selected from: (i)anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) theanthocyanin synthase has an amino acid sequence at least 80% identicalto SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69;(iii) the anthocyanin synthase has an amino acid sequence at least 80%identical to SEQ. ID NO: 13; and (iv) any combinations thereof. Incertain embodiments, one or more engineered host cells comprisesflavonoid-3-glucosyl transferase (3GT). In certain embodiments,flavonoid-3-glucosyl transferase is selected from: (i)flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii)the flavonoid-3-glucosyl transferase has an amino acid sequence at least80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ.ID NO: 73; and (iii) any combinations thereof. In certain embodiments,one or more genetic modifications comprises overexpression ofanthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). Incertain embodiments, the one or more genetic modifications comprisesoverexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more geneticmodifications are selected from a group consisting of: (i) anthocyaninsynthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) acombination thereof.

In another aspect, the invention provides a method of increasing thetransformation of leucocyanidin or catechin to cyanidin-3-glucoside(Cy3G), delphinidin or gallocatechin to delphindin-3-glucoside (De3G),or afzelechin or pelargonidin to pelargonidin-3-glucoside (Pe3G)comprising anthocyanin synthase, wherein the anthocyanin synthase isselected from: (i) anthocyanin synthase of Carica papaya (SEQ. IDNO:13); (ii) the anthocyanin synthase has an amino acid sequence atleast 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68,or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acidsequence at least 80% identical to SEQ. ID NO: 13; and (iv) anycombinations thereof. In certain embodiments, one or more geneticmodifications comprises overexpression of anthocyanin synthase andflavonoid-3-glucosyl transferase (3GT). In certain embodiments, the oneor more genetic modifications are selected from a group consisting of:(i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT),and (iii) a combination thereof.

In another aspect, the invention provides a method of increasing thetransformation of cyanidin to cyanidin-3-glucoside (Cy3G), delphindin todelphindin-3-glucoside (De3G), or pelargonidin topelagonidin-3-glucoside (Pe3G), comprising flavonoid-3-glucosyltransferase (3GT), wherein the flavonoid-3-glucosyl transferase isselected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca(SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an aminoacid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71,SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof.

In another aspect, the invention provides an engineered host cellcomprises one or more genetic modifications to increase the productionof dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL),and/or pentahydroxyflayaone (PHF), wherein the engineered host cellcomprises cytochrome P450 reductase (CPR) and at least one offlavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), orflavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, theprecursor for increase in production of dihydroquercetin (DHQ),dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone(PHF) is naringenin and/or dihydrokaempferol (DHK). In certainembodiments, the engineered host cell further comprises peptidesselected from a group consisting of: (i) flavonoid 3′-hydroxylase(F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combinationthereof. In certain embodiments, the engineered host cell produceseriodictyol or taxifolin. In certain embodiments, the engineered hostcell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certainembodiments, the engineered host cell produces pentahydroxyflavone ordihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase(F3′H) is truncated to remove the N-terminal leader sequence. In certainembodiments, cytochrome P450 reductase (CPR) is truncated to remove theN-terminal leader sequence. In certain embodiments, flavonoid3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). Incertain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused withcytochrome P450 reductase (CPR). In certain embodiments,flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO. 7. In certainembodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequenceat least 80% identical to the polypeptide set forth in SEQ ID NO. 8. Incertain embodiments, cytochrome P450 reductase (CPR) has an amino acidsequence at least 80% identical to the polypeptide set forth in SEQ IDNO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) hasan amino acid sequence at least 80% identical to the polypeptidesselected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO.56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered hostcell further comprises cytochrome b₅. In certain embodiments, cytochromeb₅ has an amino acid sequence at least 80% identical to the polypeptideset forth in SEQ ID NO. 98. In certain embodiments, wherein theflavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80%identical to the polypeptides selected from a group consisting of: (i)SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO.47, and (v) SEQ ID NO. 48.

In another aspect, the invention provides method of increasing theproduction of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl(EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered hostcell, wherein the engineered host cell comprises cytochrome P450reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H),flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase(F3′5′H). In certain embodiments, the precursor for increase inproduction of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl(EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/ordihydrokaempferol (DHK). In certain embodiments, the engineered hostcell further comprises peptides selected from a group consisting of: (i)flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR);and (iii) any combination thereof. In certain embodiments, theengineered host cell produces eriodictyol or taxifolin. In certainembodiments, the engineered host cell further comprises flavonoid3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered hostcell produces pentahydroxyflavone or dihydromyricetin. In certainembodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove theN-terminal leader sequence. In certain embodiments, cytochrome P450reductase (CPR) is truncated to remove the

N-terminal leader sequence. In certain embodiments, flavonoid3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). Incertain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused withcytochrome P450 reductase (CPR). In certain embodiments,flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO. 7. In certainembodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequenceat least 80% identical to the polypeptide set forth in SEQ ID NO. 8. Incertain embodiments, cytochrome P450 reductase (CPR) has an amino acidsequence at least 80% identical to the polypeptide set forth in SEQ IDNO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) hasan amino acid sequence at least 80% identical to the polypeptidesselected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO.56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered hostcell further comprises cytochrome b₅. In certain embodiments, cytochromeb₅ has an amino acid sequence at least 80% identical to the polypeptideset forth in SEQ ID NO. 98. In certain embodiments, wherein theflavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80%identical to the polypeptides selected from a group consisting of: (i)SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO.47, and (v) SEQ ID NO. 48.

VI. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 shows the metabolic pathway of flavonoid and anthocyaninbioproduction in engineered cells and methods of preparing anthocyaninsdescribed herein.

FIG. 2 shows structures of the flavonoid and anthocyanin molecules thatmay be produced using engineered cells and methods of preparinganthocyanins described herein.

FIG. 3 shows HPLC spectra showing peaks corresponding to the moleculesprepared using engineered cells and methods of preparing anthocyaninsdescribed herein.

FIG. 4 shows the pathway of flavonoid and anthocyanin bioproduction inengineered cells and methods of preparing anthocyanins described herein.

VII. DETAILED DESCRIPTION OF THE INVENTION

The present application provides engineered cells for producing one ormore flavonoids, cultures that include the engineered cells, and methodsof producing one or more flavonoids, or at least one anthocyanin. Theterms “flavonoid”, “flavonoid product”, or “flavonoid compound” are usedherein to refer to a member of a diverse group of phytonutrients foundin almost all fruits and vegetables. As used herein, the terms“flavonoid”, “flavonoid product”, or “flavonoid compound” are usedinterchangeably to refer a molecule containing the general structure ofa 15-carbon skeleton, which consists of two phenyl rings (A and B) and aheterocyclic ring. Flavonoids may include, but are not limited to,isoflavone type (e.g., genistein), flavone type (e.g., apigenin),flavonol type (e.g., kaempferol), flavanone type (e.g., naringenin),chalcone type (e.g., phloretin), anthocyanidin type (e.g., cyanidin),catechins, flavanones, and flavanonols. Flavonoid compounds of interestinclude, without limitation, naringenin, naringenin chalcone,eriodictyol, taxifolin, dihydrokaempferol, dihydroquercetin,dihydromyricetin, leucocyanidin, leucopelargonidin, leucodelphindin,pentahydroxyflavone, cyanidin, catechin, delphinidin, pelargonidin, andkaempferol. Anthocyanins are in the forms of anthocyanidin glycosidesand acylated anthocyanins. Anthocyanin compounds of interest include,without limitation, cyanidin glycoside, delphinidin glycoside,pelargonidin glycoside, peonidin glycoside, and petunidin glycoside.

The terms ‘precursor’ or ‘flavonoid precursor’ as used herein may referto any intermediate present in the biosynthetic pathway that leads tothe production of catechins or anthocyanins. flavonoid precursors mayinclude, but are not limited to tyrosine, phenylalanine, coumaric acid,p-coumaroyl-CoA, malonyl-CoA, pyruvate, acetyl-CoA, and naringenin.

Cells engineered for the production of a flavonoid or an anthocyanin canhave one or multiple modifications, including, without limitation, thedownregulation, disruption, or deletion of endogenous genes, theupregulation of an endogenous gene, and the introduction of exogenousgenes.

The term “non-naturally occurring”, when used in reference to an enzymeis intended to mean that nucleic acids or polypeptides include at leastone genetic alteration not normally found in a naturally occurringpolypeptide or nucleic acid sequence. Naturally occurring nucleic acids,and polypeptides can be referred to as “wild-type” or “original”. A hostcell, organism, or microorganism that includes at least one geneticmodification generated by human intervention can also be referred to as“non-naturally occurring”, “engineered”, “genetically engineered,” or“recombinant”.

A host cell, organism, or microorganism engineered to express oroverexpress a gene or nucleic acid sequence, or to overexpress an enzymeor polypeptide has been genetically engineered through recombinant DNAtechnology to include a gene or nucleic acid sequence that does notnaturally encode the enzyme or polypeptide or to express an endogenousgene at a level that exceeds its level of expression in a non-alteredcell. As nonlimiting examples, a host cell, organism, or microorganismengineered to express or overexpress a gene or a nucleic acid sequence,or to overexpress an enzyme or polypeptide can have any modificationsthat affect a coding sequence of a gene, the position of a gene on achromosome or regulatory elements associated with a gene. Overexpressionof a gene can also be by increasing the copy number of a gene in thecell or organism. Similarly, a host cell, organism, or microorganismengineered to under-express or to have reduced expression of a gene,nucleic acid sequence, or to under-express an enzyme or polypeptide canhave any modifications that affect a coding sequence of a gene, theposition of a gene on a chromosome or regulatory elements associatedwith a gene. Specifically included are gene disruptions, which includeany insertions, deletions, or sequence mutations into or of the gene ora portion of the gene that affect its expression or the activity of theencoded polypeptide. Gene disruptions include “knockout” mutations thateliminate expression of the gene. Modifications to under-express a genealso include modifications to regulatory regions of the gene that canreduce its expression.

The term “exogenous” or “heterologous” is intended to mean that thereferenced molecule or the referenced activity is introduced into thehost microbial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material that may be introduced on a vehicle such as a plasmid.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is naturally present in the host.

Genes or nucleic acid sequences can be introduced stably or transientlyinto a host cell using techniques well known in the art including, butnot limited to, conjugation, electroporation, chemical transformation,transduction, and transfection. Optionally, for exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. Furthermore, genes can be subjected to codon optimization withtechniques well known in the art to achieve optimized expression of theproteins.

The percent identity (% identity) between two sequences is determinedwhen sequences are aligned for maximum homology. Algorithms well knownto those skilled in the art, such as Align, BLAST, Clustal Omega, andothers compare and determine a raw sequence similarity or identity, andalso determine the presence or significance of gaps in the sequencewhich can be assigned a weight or score. Such algorithms also are knownin the art and are similarly applicable for determining nucleotide oramino acid sequence similarity or identity and can be useful inidentifying orthologs of genes of interest. Additional sequences addedto a polypeptide sequence, such as but not limited to immunodetectiontags, purification tags, localization sequences (presence or absence),etc., do not affect the % identity.

A homolog is a gene or genes that have the same or identical functionsin different organisms. Genes that are orthologous can encode proteinswith sequence similarity of about 45% to 100% amino acid sequenceidentity, and more preferably about 60% to 100% amino acid sequenceidentity. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Paralogs are genes related by duplication within a genome,and can evolve new functions, even if these are related to the originalone.

An engineered cell for producing flavonoids include an exogenous nucleicacid sequence encoding tyrosine ammonia lyase (TAL) activity(alternatively or in addition, an exogenous nucleic acid encodingphenylalanine ammonia-lyase (PAL) activity and an exogenous nucleic acidencoding cinnamate-4-hydroxylase (C4H) activity), an exogenous nucleicacid sequence encoding 4-coumarate-CoA ligase (4CL) activity, anexogenous nucleic acid sequence encoding chalcone synthase (CHS)activity, and an exogenous nucleic acid sequence encoding chalconeisomerase (CHI) activity. Optionally, the engineered cell can furtherinclude an exogenous nucleic acid sequence encoding an exogenous nucleicacid sequence encoding flavanone-3-hydroxylase (F3H) activity, anexogenous nucleic acid sequence encoding flavonoid 3′-hydroxlase (F3′H)activity or flavonoid 3′,5′-hydroxylase (F3′5′H), an exogenous nucleicacid sequence encoding cytochrome P450 reductase (CPR) activity, anexogenous nucleic acid sequence encoding dihydroflavonol-4-reductase(DFR) activity, and/or an exogenous nucleic acid sequence encodingleucoanthocyanidin reductase (LAR) activity.

Tyrosine ammonia-lyase (TAL) can be, for example, a member of thearomatic amino acid deaminase family that catalyzes the elimination ofammonia from L-tyrosine to yield p-coumaric acid. An exemplary tyrosineammonia lyase is the Saccharothrix espanaensis tyrosine ammonia lyase(TAL; SEQ ID NO: 1). Also considered for use in the engineered cellsprovided herein are TALs with SEQ ID NOS: 23-26, TALs listed in Table 1,TAL homologs and variants having at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity to SEQ ID:1 that have the activity of atyrosine ammonia lyase that produces p-coumaric acid from tyrosine.

TABLE 1 Tyrosine ammonia-lyase Organism GenBank Accession NumberRhodotorula glutini AGZ04575.1 Flavobacterium johnsoniae WP_012023194.1Herpetosiphon aurantiacus ABX02653.1 Rhodobacter capsulatus ADE83766.1Saccharothrix espanaensis AKE50820.1 Trichosporon cutaneum AKE50834.1

Similar to tyrosine ammonia-lyase, phenylalanine ammonia-lyase (PAL) canbe a member of the aromatic amino acid deaminase family that catalyzesthe non-oxidative deamination of L-phenylalanine to form trans-cinnamicacid. An exemplary phenylalanine ammonia-lyase is the Brevibacilluslaterosporus phenylalanine ammonia-lyase (PAL; SEQ ID NO :2). Alsoconsidered for use in the engineered cells provided herein are PALs withSEQ ID NOS: 27-29, PAL homologs and variants having at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 thathave the activity of a phenylalanine ammonia lyase that producestrans-cinnamic acid from phenylalanine.

Cinnamate-4-hydroxylase (C4H) belongs to the cytochrome P450-dependentmonooxygenase family and catalyzes the formation of p-coumaric acid fromtrans-cinnamic acid. Considered for use in the engineered cells providedherein are C4H of Helianthus annuus L. (C4H; SEQ ID NO: 3), C4Hs withSEQ ID NOS: 30-32, and C4H homologs of other species, as well asvariants of naturally occurring C4Hs having at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% amino acid identity to the SEQ ID NO: 3 (C4H,Helianthus annuus L.) that have the activity of a C4H.

4-coumarate-CoA ligase (4CL) catalyzes the activation of 4-coumarate toits CoA ester. Considered for use in the engineered cells providedherein are 4CLs of Petroselinum crispum (SEQ ID NO: 4), 4CLs in Table 2,4CLs with SEQ ID NOS: 33-36, and 4CL homologs of other species, as wellas variants of naturally occurring 4CLs having at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% amino acid identity to SEQ ID No: 4(4CL, Petroselinum crispum) that have the activity of a 4CL.

TABLE 2 4-coumarate-CoA ligases Organism GenBank Accession NumberPetroselinum crispum CAA31697.1 Camellia sinensis ASU87409.1 Capsicumannuum KAF3620173.1 Castanea mollissima KAF3954751.1 Daucus carotaAIT52344.1 Gynura bicolor BAJ17664.1 Ipomoea purpurea AHJ60263.1Lonicera japonica AGE10594.1 Lycium chinense QDL52638.1 Nelumbo nuciferaXP_010265453.1 Nyssa sinensis KAA8540582.1 Solanum lycopersicumNP_001333770.1 Striga asiatica GER48539.1

The chalcone synthase (CHS) can be, for example, a type III polyketidesynthase that sequentially condenses three molecules of malonyl-CoA withone molecule of p-coumaryol-CoA to produce the naringenin precursornaringenin chalcone or naringenin. An exemplary chalcone synthase is thechalcone synthase of Petunia x hybrida (CHS, SEQ ID NO: 5). Alsoconsidered for use in the engineered cells provided herein are the geneslisted in Table 3, CHSs with SEQ ID: 37-40, and CHS homologs andvariants having at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%amino acid identity to SEQ ID NO: 5 (CHS, Petunia x hybrida) that havethe activity of a chalcone synthase.

TABLE 3 Chalcone synthases Organism GenBank Accession Number Petuniahybrida AAF60297.1 Acer palmatum AWN08245.1 Callistephus chinensisCAA91930.1 Camellia japonica BAI66465.1 Capsicum annuum XP_016566084.1Coffea arabica XP_027118978.1 Curcuma alismatifolia ADP08987.1Dendrobium catenatum ALE71934.1 Garcinia mangostana ACM62742.1 Iochromacalycinum AIY22758.1 Iris germanica BAE53636.1 Lilium speciosumBAE79201.1 Lonicera caerulea ALU09326.1 Lycium ruthenicum ATB56297.1Magnolia liliiflora AHJ60259.1 Matthiola incana BBM96372.1 Morus albavar. multicaulis AHL83549.1 Nelumbo nucifera NP_001305084.1 Nyssasinensis KAA8548459.1 Paeonia lactiflora AEK70334.1 Panax notoginsengQKV26463.1 Ranunculus asiaticus AYV99476.1 Rosa chinensis AEC13058.1Theobroma cacao XP_007032052.2

Chalcone isomerase (CHI, also referred to as chalcone flavononeisomerase) catalyzes the stereospecific and intramolecular isomerizationof naringenin chalcone into its corresponding (2S)-flavanones.Considered for use in the engineered cells provided herein are CHI ofMedicago sativa (SEQ ID NO: 6), CHI of Table 4, CHIs with SEQ ID NOS:41-44, and CHI homologs of other species, as well as variants ofnaturally occurring CHI having at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% amino acid identity to SEQ ID NO: 6 (CHI, Medicago sativa)that have the activity of a chalcone isomerase.

TABLE 4 Chalcone Isomerases Organism GenBank Accession Number Medicagosativa AGZ04578.1 Dendrobium hybrid cultivar AGY46120.1 Abrusprecatorius XP_027366189.1 Antirrhinum majus BA032070.1 Arachisduranensis XP_015942246.1 Astragalus membranaceus ATY39974.1 Camelliasinensis XP_028119616.1 Castanea mollissima KAF3958409.1 Cephalotusfollicularis GAV77263.1 Clarkia gracilis subsp. QPF47150.1 sonomensisDianthus caryophyllus CAA91931.1 Glycyrrhiza uralensis AXO59749.1Handroanthus impetiginosus PIN05040.1 Lotus japonicus CAD69022.1 Morusalba AFM29131.1 Phaseolus vulgaris XP_007142690.1 Punica granatumANB66204.1 Rhodamnia argentea XP_030524476.1 Spatholobus suberectusTKY50621.1 Trifolium subterraneum GAU12132.1

A nucleic acid sequence encoding a CHI can in some embodiments be fusedto a nucleic acid sequence encoding a CHS in an engineered cell asprovided herein, such that the CHI activity is fused to the chalconesynthase activity, i.e., a fusion protein is produced in the engineeredcell that has both condensing and cyclization activities.

Flavanone 3-hydroxylase (F3H) catalyzes the stereospecific hydroxylationof (2S)-naringenin to form (2R,3R)-dihydrokaempferol. Other substratesinclude (2S)-eriodictyol, (2S)-dihydrotricetin and (2S)-pinocembrin.Some F3H enzymes are bifunctional and also catalyzes as flavonolsynthase (EC: 1.14.20.6). Considered for use in the engineered cellsprovided herein are F3H of Rubus occidentalis (SEQ ID NO: 7), F3Hs withSEQ ID NOS: 45-48, F3Hs listed in Table 5, and other F3H homologs andvariants having at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%amino acid identity to SEQ ID NO:7 (F3H, Rubus occidentalis) that havethe activity of a F3H.

TABLE 5 Flavanone 3-hydroxylases Organism GenBank Accession Number Rubusoccidentalis ACM17897.1 Abrus precatorius XP_027347564.1 Nyssa sinensisKAA8547483.1 Camellia sinensis AAT68774.1 Morelia rubra KAB1219056.1Rosa chinensis PRQ47414.1 Malus domestica AAD26206.1 Vitis amurensisALB75302.1 Iochroma ellipticum AMQ48669.1 Hibiscus sabdariffa ALB35017Cephalotus follicularis GAV71832

Flavonoid 3′-hydroxylases (F3′H) belongs to the cytochrome P450 familywith systematic name of flavonoid, NADPH:oxygen oxidoreductase(3′-hydroxylating). In the flavonoid biosynthetic pathway, F3′H convertsdihydrokaempferol to dihydroquercetin (taxifolin) or naringenin toeriodictyol. Considered for use in the engineered cells provided hereinare F3′H of Brassica napus (F3′H; SEQ ID NO: 8), F3′H with SEQ ID NOS:49-52, those listed in Table 6, and homologs and variants having atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% amino acididentity to these F3′H. F3′H is a cytochrome P450 enzyme that requires acytochrome P450 reductase (CPR) to function. Cytochrome P450 reductasesare diflavin oxidoreductases that supply electrons to F3′Hs. The P450reductase can be from the same species as F3′H or different species fromF3′H. Considered for use in the engineered cells provided herein are CPRof Catharanthus roseus (SEQ ID NO: 9), additional CPRs listed in Table7, CPRs with SEQ ID NOS: 53-55, CPR homologs of other species, andvariants of naturally occurring CPRs having at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%,atleast 98%, or at least 99% amino acid identity to these CPRs that havethe activity of a CPR. In various embodiments, the N-terminal nucleicacid sequences in the genes of F3′H and/or CPR originated fromeukaryotic cells can encode targeting leader peptides, which can beremoved before introduction into prokaryotic host cells, if desired. Insome embodiments, the hydroxylase complex HpaBC from E. coli was used tohydroxylate naringenin to eriodictyol or dihydrokaempferol todihydroquercetin (taxifolin).

TABLE 6 Flavonoid 3′-hydroxylases Organism GenBank Accession NumberBrassica napus ABC58722.1 Gerbera hybrid cultivar D1 ABA64468.1Cephalotus follicularis GAV84063.1 Theobroma cacao XP_007037548.1Phoenix dactylifera XP_008791304.2

TABLE 7 Cytochrome P450 reductases Organism GenBank Accession NumberCatharanthus roseus CAA49446.1 Brassica napus XP_013706600.1 Cephalotusfollicularis GAV59576.1 Camellia sinensis XP_028084858.1

A nucleic acid sequence encoding a F3′H can in some embodiments be fusedto a nucleic acid sequence encoding a CPR in an engineered cell asprovided herein, such that the F3′H activity is fused to the CPRactivity.

In the cells engineered to produce dihydomyricetin, flavonoid 3′,5′-hydroxylase (F3′5′H) can be used to convert dihydrokaempferol todihydromyricetin or naringenin to pentahydroxyflavone, which is furtherconverted to dihydromyricetin by a F3H. F3′5′H has the systematic nameflavanone, NADPH:oxygen oxidoreductase and catalyzes the formation of3′,5′-dihydroxyflavanone from flavanone. An exemplary F3′5′H is theDelphinium grandiflorum F3′5′H (SEQ ID NO: 10), Also considered for usein the engineered cells provided herein include F3′5′H with SEQ IDNOS:56-57, F3′5′H homologs of other species, and variants of naturallyoccurring F3′5′H having at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% amino acid identity to SEQ ID NOS:10 that have the activity ofa F3′5′H.

Dihydroflavonol 4-reductase (DFR) acts on (+)-dihydrokaempferol (DHK),(+)-dihydroquercetin (Taxifolin, DHQ), or dihydromyricein (DHM) toreduce those compounds to the corresponding cis-flavan-3,4-diol (DHK toleucopelargonidin; Taxifolin to leucocyanidin; DHM to leucodelphinidin).An exemplary DFR is the Anthurium andraeanum DFR (SEQ ID NO: 11). Alsoconsidered for use in the engineered cells provided herein include DFRsin Table 8, DFRs with SEQ ID NOS: 58-61, and DFR homologs of otherspecies, as well as variants of naturally occurring DFR having at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% amino acid identity toSEQ ID NO: 11.

TABLE 8 Dihydroflavonol 4-reductases Organism GenBank Accession NumberEustoma grandiflorum BAD34461.1 Anthurium andraeanum AAP20866.1 Camelliasinensis AAT66505.1 Morelia rubra KAB1203810.1 Dendrobium moniliformeAEB96144.1 Fragaria × ananassa AHL46451.1 Rosa chinensis XP_024167119.1Acer palmatum AWN08247.1 Nyssa sinensis KAA8531902.1 Vitis amurensisI82380.1 Abrus precatorius XP_027329642.1 Angelonia angustifoliaAHM27144.1 Pyrus pyrifolia Q84KP0.1 Theobroma cacao XP_017985307Theobroma cacao XP_007051597.2 Brassica oleracea var. capitataQKO29328.1 Rubus idaeus AXK92786.1 Citrus sinensis AAY87035.1 Gerberahybrida P51105.1 Cephalotus follicularis GAV76940.1 Ginkgo bilobaAGR34043.1 Dryopteris erythrosora QFQ61498.1 Dryopteris erythrosoraQFQ61499.1 Cephalotus follicularis GAV76942.1

Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechinfrom 3,4-cis-leucocyanidin. LAR also synthesizes afzelechin andgallocatechin. Considered for use in the engineered cells providedherein are LAR of Desmodium uncinatum (SEQ ID NO: 12), LARs with SEQ IDNOS: 62-65, and LAR homologs of other species, as well as variants ofnaturally occurring LAR having at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% amino acid identity to SEQ ID NO: 12 (LAR, Desmodiumuncinatum) that have the activity of a LAR.

Optionally, the cells are further engineered to include an anthocyaninsynthase (ANS) which catalyzes the conversion of leucoanthocyanidin orcatechin to anthocyanidin, leucopelargonidin to pelargonidin, orleucodelphinidin to delphinidin. Considered for use in the engineeredcells provided herein are ANS of Carica papaya (SEQ ID NO: 13), ANS withSEQ ID NOS: 66-69, and ANS homologs of other species, as well asvariants of naturally occurring ANS having at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% amino acid identity to SEQ ID NO:13 (ANS,Carica papaya) that have the activity of a ANS.

Optionally, the cells are further engineered to include aflavonoid-3-glucosyl transferase (3GT) to generate anthocyanins bytransfer of a sugar moiety such as, without limitation, UDP-α-D-glucoseto anthocyanidins to form glycosylated anthocyanins. Considered for usein the engineered cells provided herein are 3GT of Vitis labrusca (SEQID NO:14), 3GT with SEQ ID NOS: 70-73, and 3GT homologs of otherspecies, as well as variants of naturally occurring 3GT having at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% amino acid identity toSEQ ID NO: 14 (3GT, Vitis labrusca) that have the activity of a 3GT.

In various aspects, host cells may be engineered for enhanced productionof flavonoids or anthocyanins by introducing additional exogenouspathways and/or modifying endogenous metabolic pathways to remove ordownregulate competitive pathways to reduce carbon loss, increaseprecursor supply, improve cofactor availability, reduce byproductformation, or improve cell fitness. Enhancing or improving production offlavonoids or anthocyanins can be increasing yield, titer, or rate ofproduction.

Thus, a host cell engineered for the production of a flavonoid oranthocyanin can be engineered to include any or any combination of:overexpression of an acetyl-CoA carboxylase (ACC) or an ACC variant;expression or overexpression of at least one enzyme for increasingcell's malonyl-CoA supply that does not rely on the ACC step; expressionor overexpression of at least one enzyme to increase tyrosine supply;expression or overexpression of at least one enzyme to increase CoAavailability for synthesizing precursors malonyl-CoA or p-coumaryol-CoA;expression or overexpression at least one enzyme to increase hemebiosynthesis; deletion or downregulation of at least one fatty acidsynthesis enzyme; at least one alcohol dehydrogenase, lactatedehydrogenase, pyruvate oxidase, phosphate acetyl transferase, oracetate kinase; at least one enzyme of a fatty acid degradation pathway,at least one thioesterase, or at least one TCA gene. The foregoing listof modifications is nonlimiting.

Malonyl-CoA is the direct precursor for chalcone synthase to performsequential condensations with p-coumaryol-CoA. Malonyl-CoA supply can beincreased by one or more modifications. Malonyl-CoA is synthesized byacetyl-CoA carboxylase (ACC) via the ATP-dependent carboxylation ofacetyl-CoA in a multistep reaction. First, the biotin carboxylase domaincatalyzes the ATP-dependent carboxylation of biotin using bicarbonate asa CO₂ donor. In the second reaction, the carboxyl-group is transferredfrom biotin to acetyl-CoA to form malonyl-CoA. In most eukaryotes,including fungi, both reactions are catalyzed by a large single chainprotein, but in E. coli and other bacteria, the activity is catalyzed bya multi-subunit enzyme. Host cells can be engineered for example toexpress an exogenous acetyl-CoA carboxylase or a variant ACC to increasemalonyl-CoA synthesis from acetyl-CoA. For example, Mucor circinelloides(SEQ ID NO: 15) acetyl-CoA carboxylase can be introduced into the hostcells. Additional examples of ACC genes that may be used in theengineered cells provided herein include, without limitation, the geneslisted in Table 9, genes with SEQ ID NOS: 74-76, naturally occurringorthologs of these ACCs, or variants having at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% amino acid identity to referenced genes.Further, naturally occurring acetyl-CoA carboxylase genes can be furtherengineered to introduce single or multiple amino acid mutations toincrease catalytic activity and/or remove feedback inhibition.

TABLE 9 Acetyl-CoA carboxylases Organism GenBank Accession NumberLipomyces starkeyi AJT60321.1 Rhodotorula toruloides GEM08739.1 Ustilagomaydis XP_011390921.1 Mucor circinelloides EPB82652.1 Kalaharituberpfeilii KAF8466702.1 Aspergillus fumigatus KEY77072.1 Rhodotoruladiobovata TNY18634.1 Leucosporidium creatinivorum ORY74050.1Microbotryum intermedium SCV70467.1 Mixia osmundae GAA98306.1 Pucciniagraminis KAA1079218.1 Suillus occidentalis KAG1764021.1 Gymnopilusjunonius KAF8909366.1

Additional strategies for increasing malonyl-CoA include increasingacetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase(ACC). Acetyl-CoA can be synthesized from acetate by an acyl-CoA ligasein an ATP-dependent reaction. Acetyl-CoA synthetase (ACS) or acetate-CoAligase (EC 6.2.1.1.) catalyzes the formation of a new chemical bondbetween acetate and CoA coenzyme A (CoA). ACSs with native activity onacetate will provide the function of increasing acetyl-CoA supply whencells are either supplied with acetate as a co-feed, or where acetate isproduced as a by-product. Other acyl-CoA ligases, having their mainactivity on other acid substrates, may also have substantial activity onacetate, and are viable candidates for providing acetate-CoA ligaseactivity in the engineered cells provided herein. The ACSs expressed inthe host cells can be prokaryotic or eukaryotic. Cultures of engineeredhost cells that overexpress a nucleic acid sequence encoding ACS canoptionally include acetate in the culture medium. Examples of acetyl-CoAsynthase that can be expressed in a host cell engineered to produce aflavonoid or anthocyanin include, without limitation, the ACS gene of E.coli, the ACS of Salmonella typhimurium (SEQ ID NO:16), and orthologs ofthese ACSs in other species having at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% amino acid identity to these ACSs.

Alternatively, or in addition, an engineered host cell can overexpress agene encoding pyruvate dehydrogenase (PDH), which converts pyruvate toacetyl-CoA, to increase acetyl-CoA supply. PDH catalyzes an irreversiblemetabolic step, and the control of its activity is complex and involvescontrol by its substrates and products. Nicotinamide adeninedinucleotide hydrogen (NADH), a product of the PDH reaction, is acompetitive inhibitor of the PDH complex. The NADH sensitivity of thePDH complex has been demonstrated to reside in LPD, the enzyme thatinteracts with NAD+ as a substrate. Thus, a variant of the Lpd subunitof PDH can be expressed that includes one or more mutations that reducesinhibition of PDH by NADH. Such an example is a LPD variant in E. colithat contains E354K mutation, and the mutated enzyme was less sensitiveto NADH inhibition than the native LPD.

Alternatively, or in addition to strategies for increasing ACC activityand strategies for increasing acetyl-CoA, strategies for increasingmalonyl-CoA by mechanisms that do not rely on the activity of an ACC canbe employed. For example, a cell engineered to produce a flavonoid or ananthocyanin as provided herein can include an exogenous nucleic acidsequence encoding a malonyl-CoA synthetase (EC 6.2.1.14) that generatesmalonyl-CoA from malonate. Acyl-CoA synthetase catalyzes the conversionof a carboxylic acid to its acyl-CoA thioester through an ATP-dependenttwo-step reaction. In the first step, the free fatty acid is convertedto an acyl-AMP intermediate with the release of pyrophosphate. In thesecond step, the activated acyl group is coupled to the thiol group ofCoA, releasing AMP and the acyl-CoA product. Nonlimiting examples ofmalonyl-CoA synthetases include the malonyl-CoA synthetases ofStreptomyces coelicolor (SEQ ID NO:17), matB of Rhodopseudomonaspalustris (SEQ ID NO: 77), matB of Rhizobium sp, BUS003 (SEQ ID NO: 78),matB of Ochrobacrum sp. (SEQ ID NO: 79), or other homologs having atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to thereferenced sequences. Malonate can optionally be added to the culturemedium of a culture that includes a cell engineered to express amalonyl-CoA synthetase. In Rhizobium trifolii, the math gene is part ofthe matABC operon, with matA encoding a malonyl-CoA decarboxylase andmatC encoding a putative dicarboxylate carrier protein or malonatetransporter. An engineered cell that includes an exogenous gene encodinga malonyl-CoA synthetase can also include an exogenous nucleic acidsequence encoding a malonate transporter, such as a malonate transporterencoded by a matC gene, for example of Streptomyces coelicolor (SEQ IDNO:18), of Rhizobiales bacterium (SEQ ID NO:80), of Rhizobiumleguminosarum (SEQ ID NO:81), of Agrobacterium vitis (SEQ ID NO: 82), ofNeorhizobium sp. (SEQ ID NO: 83), or a malonate transporter encoded byDctPQM of Sinorhizobium medicae, or encoding a malonyl-CoA transporterhaving at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identityto a naturally-occurring malonate transporter. Cell cultures of a hostcell engineered to express a malonyl-CoA synthetase and a malonatetransporter can include a culture medium that includes malonate.

In additional embodiments, a cell engineered to produce a flavonoid oran anthocyanin is further engineered to include an exogenous nucleicacid sequence encoding malonate CoA-transferase (EC:2.8.3.3; alsoreferred to as the alpha subunit of malonate decarboxylase) that makesmalonyl-CoA by direct transfer of the CoA from acetyl-CoA. For example,the alpha subunit of malonate decarboxylase from the mdcACDE genecluster in Acinetobacter calcoaceticus has the malonate CoA-transferaseactivity. The mdcA gene product, the α subunit, is malonateCoA-transferase, and mdcD gene product, the β subunit, is a malonyl-CoAdecarboxylase. The mdcE gene product, the γ subunit, may play a role insubunit interaction to form a stable complex or as a codecarboxylase.The mdcC gene product, the δ subunit, was an acyl-carrier protein, whichhas a unique CoA-like prosthetic group. When the α subunit is removedfrom the complex and incubated with malonate and acetyl-CoA, theacetyl-CoA moiety of the prosthetic group binds on an a subunit toexchange the acetyl group for a malonyl group. As the thioester transfershould be thermodynamically favorable, the engineered cells can includea nucleic acid encoding a malonate CoA-transferase to increasemalonyl-CoA supply. Examples of mdcAs that can be expressed in anengineered cell as provided herein include, without limitation, mdcA ofAcinetobacter calcoaceticus (SEQ ID NO: 19), mdcAs of Table 10, mdcAswith SEQ ID NOS: 84-87, or a transferase having at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity to any of these or othernaturally occurring malonate CoA-transferases.

TABLE 10 Malonate CoA-transferases (malonate decarboxylase subunitalpha) Organism GenBank Accession Number Acinetobacter calcoaceticusAAB97627.1 Geobacillus sp. QNU36929.1 Acinetobacter johnsoniiWP_087014029.1 Acinetobacter marinus WP_092618543.1 Acinetobacter rudisWP_016655668.1 Psychrobacter sp. G WP_020444454.1 Moraxella catarrhalisWP_064617969.1 Zoogloea sp. MBL0283742.1 Dechloromonas sp. KAB2923906.1Stenotrophomonas rhizophila WP_123729366.1 Xanthomonas cucurbitaeWP_159407614.1

In some embodiments, a cell engineered to produce flavonoids oranthocyanins is further engineered to increase the supply of coenzyme A(CoA) to increase its availability for producing acetyl-CoA,malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoAsupply include expressing or overexpressing at least one enzyme of a CoAbiosynthesis pathway. Pantothenate kinase (EC 2.7.1.33, PanK; CoaA) isthe first enzyme in the coenzyme CoA biosynthetic pathway. Itphosphorylates pantothenate (vitamin B5) to form 4′-phosphopantothenateat the expense of a molecule of adenosine triphosphate (ATP). It is therate-limiting step in the biosynthesis of CoA. Three distinct types ofPanK have been identified—PanK-I (found in bacteria), PanK-II (mainlyfound in eukaryotes, but also in the Staphylococci) and PanK-III, alsoknown as CoaX (found in bacteria). In E. coli, pantothenate kinase iscompetitively inhibited by CoA itself, as well as by some CoA esters.The type III enzymes CoaX are not subject to feedback inhibition by CoA.In some embodiments, a host cell can be engineered to include a nucleicacid sequence encoding type III pantothenate kinase that is not feedbackinhibited by coenzyme A, such as, without limitation, CoaX gene ofPseudomonas aeruginosa (EC:2.7.1.33, SEQ ID NO: 20), CoaX ofStreptomyces sp. CL12509 (SEQ ID NO: 88), CoaX of Streptomyces cinereus(SEQ ID: 89), or CoaX of Kitasatospora kifunensis (SEQ ID NO: 90)Cultures of cells engineered for the production of flavonoids oranthocyanins can in some embodiments include a medium that includespantothenate, a precursor of CoA biosynthesis, and can optionally alsoinclude cysteine, used in the CoA biosynthesis.

Additional strategies to increase malonyl-CoA flux to the flavonoidpathway include mutation or downregulation of one or more genes thatfunction in fatty acid biosynthesis. Fatty acid biosynthesis directlycompetes with flavonoid biosynthesis for the precursor malonyl-CoA andthus limits flavonoid formation. Without limiting the embodiments to anyparticular mechanism, limiting fatty acid biosynthesis can increase themalonyl-CoA supply available for flavonoid biosynthesis. In someembodiments, the gene beta-ketoacyl-ACP synthase II (E. coli fabF) canbe disrupted, attenuated or deleted to reduce fatty acid biosynthesis.Another example of a fatty acid biosynthesis gene of a host cell thatmay be mutated or downregulated is a gene encoding malonyl-CoA-ACPtransacylase (E. coli fabD). Other fatty acid biosynthesis genes of theengineered host cell that can be downregulated include abeta-ketoacyl-ACP synthase I enzyme (E. coli fabB) and/or acyl carrierprotein (E. coli acpP).

Additional genetic modifications that may be present in a host cellengineered to produce flavonoids or anthocyanins include downregulation,disruption, or deletion of the gene targets that divert carbon flux toform byproducts such as ethanol, acetate, and lactate. They includegenes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvateoxidase, acetyl phosphate transferase and acetate kinase. In an E. colihost cell, genes that are downregulated, disrupted, or deleted caninclude adhE, ldhA, poxB, and ackA-pta.

Further, a cell engineered for the production of flavonoids oranthocyanins can have one or more genes encoding thioesterasesdownregulated, disrupted, or deleted to prevent hydrolysis of precursorsmalonyl-CoA, acetyl-CoA, and/or p-coumaryol-CoA. Acyl-CoA thioesteraseenzymes (ACOTs) catalyze the hydrolysis of acyl-CoAs (short-, medium-,long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, tothe free fatty acid and coenzyme A. For example, in an E. coli host oneor more of the thioesterase genes tesA, tesB, yciA, and/or ybgC can bedownregulated, disrupted, or deleted.

In further embodiments, a cell engineered for the production offlavonoids or anthocyanins can have one or more of fatty aciddegradation genes downregulated, disrupted, or deleted to improveprecursor supply to the flavonoid pathway. In E. coli, for example, theacyl-coenzyme A dehydrogenase (fade) gene encoding acyl-CoAdehydrogenase, adhesion A (fadA) gene encoding 3-ketoacyl-CoA thiolase,and/or gene encoding fatty acid oxidation complex subunit alpha (fadB)can be downregulated, disrupted, or deleted.

Alternatively, or in addition, genes encoding enzymes of thetricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can bedisrupted or downregulated to increase alpha-ketoglutarate supply whichserves as a cofactor for the flavonoid and anthocyanin pathway enzymes.Other TCA enzymes that can be downregulated include citrate synthasethat converts acetyl-CoA to citrate.

Also considered, in further embodiments, is an engineered host cell forthe production of flavonoids or anthocyanins to upregulate theendogenous biosynthesis of amino acid tyrosine. Tyrosine is one of theprecursors for the flavonoid biosynthesis and its conversion to4-coumaric acid is the first committed step of the pathway. Efficientbiosynthesis of L-tyrosine from feedstock such as glucose or glycerol isnecessary to make biological production economically viable. L-tyrosineis one of the three aromatic amino acids derived from the shikimatepathway. The shikimate pathway is the central metabolic route leading toformation of tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE),this pathway exclusively exists in plants and microorganisms. It startswith the condensation of intermediates of glycolysis andpentosephosphate-pathway, phosphoenolpyruvate (PEP), anderythrose-4-phosphate (E4P), respectively, which enter the pathwaythrough a series of condensation and redox reactions via3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP), 3-dehydroquinate(DHQ), 3-dehydroshikimate (DHS) to shikimate. From there the centralbranch point metabolite chorismate is obtained via shikimate-3-phosphateunder ATP hydrolysis and introduction of a second PEP. The initial stepof the shikimate pathway is catalyzed by DAHP synthase isozymes andregulated through feedback-inhibition. In E. coli three DAHP synthaseisozymes exist (aroF, aroG, aroH), which are each feedback inhibited byone of the three aromatic amino acids (TYR, PHE, TRP), in contrast thetwo DAHP synthases of plants are not subject to feedback-inhibition. Inplants and bacteria, the subsequent five steps are catalyzed by singleenzymes. From the central intermediate chorismate the pathway branchesoff to anthranilate and prephenate leading to aromatic amino acid,para-hydroxybenzoic acid (pHBA) and para-aminobenzoic acid (pABA)synthesis, the latter being a precursor for folate metabolism.Strategies to increase L-tyrosine production can include, withoutlimitation, transcriptional deregulation, removing feedback inhibition,overexpression of rate-limiting enzymes, and/or deletion of theL-phenylalanine branch of the aromatic acid biosynthetic pathway. Forexample, in an E. coli host the tyrR gene can be disrupted,feedback-inhibition-resistant versions of the DAHP synthase (aroG) andchorismate mutase (tyrA) can be introduced, and/or rate-limitingenzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimatedehydrogenase (ydiB) can be overexpressed. Further, the ppsA, aroG,and/or transketolase (tktA) can be overexpressed or exogenouslyintroduced to enhance tyrosine production.

Also considered, in further embodiments, is an engineered host cell forthe production of flavonoids or anthocyanins further engineered toupregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450(CYPs), one of the exogenous genes in the engineered cells providedherein, contain heme as a cofactor. Improving heme supply can be aneffective strategy to increase flavonoid biosynthesis. 5-aminolevulinicacid (ALA) is the first committed precursor to the heme pathway. Thereexist two known alternate routes by which this committed intermediate isgenerated. One route is the C4 pathway (Shemin pathway), which involvesthe condensation of succinyl-CoA and glycine to D-aminolevulinic acid byALA synthase (ALAS). The C4 pathway is restricted to mammals, fungi andpurple nonsulfur bacteria. The second route is the C5 pathway, whichinvolves three enzymatic reactions resulting in the biosynthesis of ALAfrom the five-carbon skeleton of glutamate. The C5 pathway is active inmost bacteria, all archaea and plants. Seven additional reactions,including assembly of eight ALA molecules into a cyclic tetrapyrrole,modification of the side chains, and incorporation of reduced iron intothe molecule, are required to convert ALA to heme. In an E. coli host,the three enzymes involved in ALA biosynthesis are glutamyl-tRNAsynthetase (G1tX), glutamyl-tRNA reductase (hemA), andglutamate-1-semialdehyde aminotransferase (hemL). In an E. coli host,the engineered cells provided herein can be further engineered toexpress or overexpress hemA or its variants, and/or hemL to increase theheme precursor ALA production. The nonlimiting examples of hemA genethat can be overexpressed include, without limitation, a mutated hemAgene from Salmonella typhimurium (EC:1.1.1.70, SEQ ID NO: 21) and hemAwith SEQ ID NOS: 91-93. Alternatively, or in addition, a heterologousALAS gene can be introduced to produce ALA via the C4 pathway.Nonlimiting examples of heterologous ALAS that can be expressed in E.coli include ALAS of Rhodobacter capsulatus (SEQ ID:22), ALAS with SEQID NOS: 94-97, or an ALAS having at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity to any of these or othernaturally-occurring ALAS. Further, one or more of the downstream genes(E. coli hemB, hemC, hemD, hemE, hemF, hemG, hemI, or hemH) thatcatalyze the synthesis of heme from ALA can be overexpressed to drivethe flux from ALA to heme production. Cultures of cells engineered forthe production of flavonoids or anthocyanins can in some embodimentsinclude a medium that includes succinate and/or glycine, precursors ofheme biosynthesis via the C4 pathway.

Engineered cells that produce a flavonoid can be engineered to includemultiple pathways to enhance flavonoid production. Those skilled in theart will recognize that the embodiments described herein can be combinedin multiple ways. Examples of engineered cells having multiple geneticmodifications are exemplary only and do not limit the scope of theinvention.

Enzymes to be expressed or overexpressed in engineered cells accordingto the invention are set forth in Table 11.

Host Cells

A host cell as provided herein can be a prokaryotic cell or a eukaryoticcell. Eukaryotic cells may be microbial eukaryotic cells, such as, forexample, fungal cells or yeast cells. Prokaryotic cells that can beengineered as provided herein include bacterial cells and cyanobacteria)cells.

Host can be selected based on their ability to take up and utilizeparticular carbon sources, nitrogen sources, or precursor molecules ormay be engineered to take up and utilize molecules that may be added tothe culture medium.

Nonlimiting examples of suitable microbial hosts for the bio-productionof a flavonoid include, but are not limited to, any gram-negativeorganisms, more particularly a member of the family Enterobacteriaceae,such as E. coli, any gram-positive microorganism, for example Bacillussubtilis, Lactobacillus sp. or Lactococcus sp.; a yeast, for exampleSaccharomyces cerevisiae, Pichia pastoris or Pichia stipitis; and othergroups or microbial species. More particularly, suitable microbial hostsfor the bio-production of a flavonoid generally include, but are notlimited to, members of the genera Clostridium, Zymomonas, Escherichia,Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter,Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, andSaccharomyces.

Culture Medium

In yet another aspect, methods for producing a flavonoid or ananthocyanin that include incubating a culture of an engineered host cellas provided herein to produce a flavonoid or an anthocyanin. The methodscan further include recovering the flavonoid or anthocyanin from theculture medium, whole culture, or cells.

The culture comprises cells engineered for the production of flavonoidsor anthocyanins in a culture medium. In various embodiments theengineered cells can be prokaryotic or eukaryotic cells. The culturemedium includes at least one carbon source that is also an energysource. Exemplary carbon sources include glucose, glycerol, sucrose,fructose, and xylose. Such carbon sources may be purified or crude,including a biomass comprising glycerol, for example, crude glycerolproduced as a byproduct of biodiesel production from corn waste. Inaddition, the culture medium can include one or more other carbonsources or compounds to increase precursor generation or cofactor supplysuch as, without limitation, tyrosine, phenylalanine, coumaric acid,acetate, malonate, succinate, glycine, bicarbonate, biotin, naringenin,5-aminolevulinic acid, thiamine, pantothenate, alpha-ketoglutarate, andascorbate. In some embodiments, tyrosine and coumaric acid are providedin the culture medium. In some embodiments, tyrosine,alpha-ketoglutarate, 5-aminolevulinic acid, and ascorbate are providedin the culture medium.

Culture conditions can include aerobic, microaerobic or any combinationalternating aerobic/microaerobic growth conditions. Further, cultureconditions can include shake flasks, fermentation, and other large scaleculture procedures. An exemplary growth condition for achieving aflavonoid product include aerobic or microaerobic fermentationconditions. The culture conditions can be scaled up and growncontinuously for manufacturing flavonoid product. Exemplary growthprocedures include, for example, fed-batch fermentation and batchseparation. In an exemplary batch fermentation protocol, the cells aregrown in a bioreactor that is well controlled for growth temperature,oxygen, pH, carbon sources, and other compounds. The desired temperaturecan be from, for example, 20-37° C., depending on the growthcharacteristics of the production cells and desired conditions for thefermented products. The pH of the bioreactor can be controlled to rangefrom 5-8 or left uncontrolled in some cases. The batch fermentationperiod can last in the range of several hours to several days, forexamples, 8 to 96 hours. Upon completion of the cultivation period, thefermenter contents can be passed through a cell separation unit toremove cells and cell debris. The cells can be lysed or disruptedenzymatically or chemically prior to or after separation of cells fromthe fermentation broth, as desired, in order to release additionalproduct. To purify the flavonoids and/or anthocyanins to homogeneity thesolution containing the flavonoids and/or anthocyanins was concentratedand the product purified via ion exchange or silica-basedchromatography. The resulting solution was either lyophilized to yieldthe products in a solid form or was concentrated into a liquid solution.

In some embodiments, a method of producing a flavonoid or an anthocyanincomprises culturing an engineered cell disclosed herein in a culturemedium to produce a flavonoid or an anthocyanin. In some embodiments,glycerol is used as a carbon feedstock. In some embodiments, theglycerol is crude glycerol. In some embodiments, the method comprisesisolating naringenin, dihydrokaempferol, taxifolin, eriodictyol,leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin,cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidinglucoside or pelargonidin glucoside. In some embodiments the naringenin,dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin,leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin,delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside orpelargonidin glucoside may be isolated at a purity of greater than 50%,greater than 55%, greater than 60%, greater than 65%, greater than 70%,greater than 75%, greater than 80%, greater than 85%, greater than 90%,or greater than 95%. In some embodiments, the naringenin,dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin,leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin,delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside orpelargonidin glucoside may be isolated at a purity of from about 50% toabout 99%, e.g., from about 50% to about 95% (for example from: about50%, 55%, 60%, 65%, 70%, 75%, 80% to about: 85%, 90%, 95%, 97.5%, 99% or99.9%). In some embodiments, the naringenin, dihydrokaempferol,taxifolin, eriodictyol, leucocyanidin, leucodelphinidin,leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin,cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside maybe isolated at a purity of from about 50% to: about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, or about 99%. In some embodiments, the naringenin,dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin,leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin,delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside orpelargonidin glucoside may be isolated at a purity of from about 55% to:about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 99%. In some embodiments, the naringenin,dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin,leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin,delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside orpelargonidin glucoside may be isolated at a purity of from about 60% to:about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, or about 99%. In some embodiments, the naringenin,dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin,leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin,delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside orpelargonidin glucoside may be isolated at a purity of from about 65% to:about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orabout 99%. In some embodiments, the naringenin, dihydrokaempferol,taxifolin, eriodictyol, leucocyanidin, leucodelphinidin,leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin,cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside maybe isolated at a purity of from about 70% to: about 75%, about 80%,about 85%, about 90%, about 95%, or about 99%. In some embodiments, thenaringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin,leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin,delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside orpelargonidin glucoside may be isolated at a purity of from about 75% to:about 80%, about 85%, about 90%, about 95%, or about 99%, from about 80%to about 85%, about 90%, about 95%, or about 99%. In some embodiments,the naringenin, dihydrokaempferol, taxifolin, eriodictyol,leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin,cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidinglucoside or pelargonidin glucoside may be isolated at a purity of fromabout 85% to: about 90%, about 95%, or about 99%. In some embodiments,the naringenin, dihydrokaempferol, taxifolin, eriodictyol,leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin,cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidinglucoside or pelargonidin glucoside may be isolated at a purity of fromabout 90% to about 95%, or about 99%, or from about 95% to about 99% orgreater.

VIII. EXAMPLES Using The Modified Cell To Create Products Example 1Production of Naringenin in E. coli

An E. coli cell derived from MG1655 was engineered to overexpress ACC(SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO:5), and CHI (SEQ ID NO: 6) to produce naringenin when substratestyrosine and coumaric acid were supplied in culture medium. ACC wasexpressed on a medium-copy plasmid (15-20 copies) while TAL, 4CL, CHS,and CHI were expressed on the chromosome. Cells of an OD 2.5 werecultured in a 48-well plate at 30 degree for 24 hours with a shakingspeed of 600 RPM in minimal medium supplied with trace element,vitamins, 1 mM tyrosine,1 mM coumaric acid, and 2% glycerol. Cellcultures were extracted with DMSO at 1:1 ratio and centrifuged for 15mins. The supernatant was analyzed for naringenin with HPLC. The cellsproduced 232 μM naringenin.

Variants of the foregoing host cell may be prepared using one or more ofACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ IDNO: 5), and CHI (SEQ ID NO: 6) with one or more homologs of ACC (SEQ IDNO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), orCHI (SEQ ID NO: 6), or combinations of two or more thereof, wherein thehomologous enzymes have at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity to the referenced enzymes.

Example 2 Production of Dihydrokaempferol in E. coli

An E. coli cell derived from MG1655 was engineered to overexpress F3H(SEQ ID NO: 7) on the chromosome to produce dihydrokaempferol whensubstrate naringenin was supplied in culture medium. Cells of an OD0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours witha shaking speed of 200 RPM in minimal medium supplied with 2% glycerol,trace elements, 0.8 mM naringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mMferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cellcultures were extracted with DMSO and centrifuged for 15 minutes. Thesupernatant was analyzed for dihydrokaempferol with HPLC. The cellsproduced 315 μM dihydrokaempferol.

Variants of the foregoing host cell may be prepared using a homolog ofF3H (SEQ ID NO: 7), wherein the homologous enzyme has at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to the referencedenzyme.

Example 3 Production of Taxifolin in E. coli

An E. coli strain derived from MG1655 was engineered to overexpress F3H(SEQ ID NO: 7), F3′H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) to producetaxifolin when the substrate naringenin was supplied in culture medium.F3H was overexpressed on the chromosome while F3′H and CPR wereoverexpressed on a medium-copy plasmid. Cells of an OD 0.5-0.7 werecultured in a 24-well plate at 30 degree for 18 hours with a shakingspeed of 200 RPM in minimal medium supplied with 2% glucose, 0.8 mMnaringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mM ferrous sulfate, 0.1 mM2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extractedwith 50% DMSO and centrifuged for 15 minutes. The supernatant wasanalyzed for taxifolin with HPLC. The cells produced 500 μM taxifolin.

Variants of the foregoing host cell may be prepared using one or more ofF3H (SEQ ID NO: 7), F3′H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) alongwith one or more homologs of F3H (SEQ ID NO: 7), F3′H (SEQ ID NO: 8),and CPR (SEQ ID NO: 9), or combinations of two or more thereof, whereinthe homologous enzymes have at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity to the referenced enzymes.

Example 4 Production of Anthocyanidins and Anthocyanins

An E. coli strain derived from MG1655 was engineered to overexpress ANS(SEQ ID NO: 13) and 3GT (SEQ ID NO: 14) to producecyanidin-3-O-glucoside when the substrate (+)-catechin was supplied inculture medium. ANS and 3GT were overexpressed on the chromosome. Cellsof an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18hours with a shaking speed of 200 RPM in minimal medium supplied with1.0% glucose, 2.0 mM (+)-catechin, 0.1 mM 2-oxoglutarate, and 2.5 mMascorbic acid. Cell cultures were acidified with 2M HCL and extractedwith 100% Ethanol. The supernatant was analyzed forcyanidin-3-O-glucoside by HPLC. The cells produced 50 mg/Lcyanidin-3-O-glucoside.

Variants of the foregoing host cell may be prepared using one or both ofANS (SEQ ID NO: 13) and 3GT (SEQ ID NO: 14) along with a homolog of ANS(SEQ ID NO: 13), 3GT (SEQ ID NO: 14), or both, wherein the homologousenzymes have at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the referenced enzymes.

Analytical Methods Example 5 Flavonoid Precursors and Flavonoids

For sampling naringenin, eriodictyol, dihydrokaempferol and taxifolin,extraction of total flavonoids from E. coli were performed on whole cellbroth. 500 μL of whole cell broth was vortexed for 30 seconds with 500μL of DMSO (dimethyl sulfoxide) and centrifuged for 15 minutes. For HPLCanalysis, 50 μL of supernatant was transferred to an HPLC vial.

The HPLC method was as follows: An Agilent 1200 HPLC was fitted with anAscentis C18 Column 150 mm×4.6 mm, 3μm, equipped with a R-18 (3 μm)guard column. The column was heated to 30 ° C. with the sample blockbeing maintained at 25 ° C. For each sample, 5 μL was injected and theproduct was eluted at a flow rate of 1.5 mL/min using 0.1% phosphoricacid in water (solvent A), acetonitrile (solvent B), and methanol(solvent C) with the following gradient:

Time A (%) B (%) C (%) 0 85 10 5 2.5 85 10 5 7.5 70 25 5 12.5 50 45 5 1585 10 5

The run time was a total of 15 minutes with naringenin, eriodictyol,dihydrokaempferol and taxifolin eluting at 12.50, 11.56, 10.20, and 8.85minutes respectively. A diode array detector (DAD) was used for thedetection of the molecule of interest at 288 nm.

Example 6 Anthocyanidins and Anthocyanins

For sampling (+)-catechin, cyanidin, and cyanidin-3-glucoside thereaction fluid was acidified with 13 M HCl (1:40 v/v), and extractedwith 100% ethanol followed by mixing, centrifugation and filtrationthrough a 0.45 μm filter. The HPLC method was as follows: An Agilent1200 HPLC was fitted with a LiChrospher RP-8 Column 250 mm×4.6 mm, 5 μm,equipped with a LiChrospher 100 RP-8 (5 μm) LiChroCART 4-4 guard column.The column was heated to 25° C. with the sample block being maintainedat 25° C. For each sample, 10 μL was injected and the product was elutedat a flow rate of 1.0 ml/min using 0.1% phosphoric acid in water(solvent A) and acetonitrile (solvent B) with the following gradient:90% A to 10% A for 12 min, 90% A for 0.5 min, and 90% A for 3.5 min forcolumn equilibration. The run time was a total of 16 minutes withcyanidin-3-glycoside eluting at 6.95 mins and cyanidin eluting at 8.9minutes. A diode array detector (DAD) was used for the detection of themolecule of interest at either 280 nm or 530 nm.

Example 7 Flavonoid Production

The example provides a combination of modifications to the E. coli hostgenome including deletions and overexpression of enzymes from otherorganisms to recapitulate the bioproduction pathway described in FIG. 4.Accordingly, the invention provides an engineered host cell thatcomprises one or more genetic modifications (as shown in FIG. 4 anddescribed in this Example 7 and herein above in this application) thatresult in production of flavonoid or anthocyanin from a carbon sourcethat can also be an energy source, through multiple chemicalintermediates, by the engineered host cell. In certain embodiments, theproduction of flavonoid or anthocyanin from a carbon source that canalso be an energy source occurs through enzymatic transformation. Incertain embodiments, the carbon source is selected from a groupconsisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv)an amino acid, and (v) any combination thereof. In certain embodiments,the engineered host cell is cultured in a medium comprising moleculesselected from a group consisting of: (i) glycerol, (ii) a sugar, (iii)an organic acid, (iv) an amino acid, and (v) any combination thereof. Asshown in FIG. 4, in certain embodiments, one or more geneticmodifications lead to increase in metabolic flux to flavonoid precursorsor cofactors. As shown in FIG. 4, in certain embodiments, one or more ofthe genetic modifications cause reduction of formation of byproducts. Asshown in FIG. 4, in certain embodiments, one or more geneticmodifications are selected from: (i) one or more modifications forover-expressing one or more endogenous genes in the engineered hostcells; (ii) one or more modifications for under-expressing one or moreendogenous genes in the engineered host cells; (iii) one or more geneticmodification is expressing one or more non-native genes in theengineered host cells; and (iv) a combination thereof.

As shown in FIG. 4, in certain embodiments, the engineered host cell iscultured in a medium comprising molecules selected from: tyrosine,phenylalanine, malonate, p-coumarate, bicarbonate, acetate,pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and5-aminolevulinic acid.

As shown in FIG. 4, in certain embodiments, the engineered host cellcomprises at least one or more nucleic acid sequences selected from: (i)a nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii)a nucleic acid sequences encoding phenylalanine ammonia lyase activity;(iii) cinnamate 4-hydroxylase; and (iv) any combination thereof. Asshown in FIG. 4, in certain embodiments, the engineered host cellcomprises at least one or more peptides selected from: (i) chalconeisomerase; (ii) chalcone synthase; (iii) a fusion protein comprises achalcone synthase and a chalcone isomerase; and (iv) any combinationthereof.

As shown in FIG. 4, in certain embodiments, one or more geneticmodifications decreases fatty acid biosynthesis. As shown in FIG. 4, incertain embodiments, the engineered host cell comprises an exogenousnucleic acid sequence selected from: (i) nucleic acid sequence encodingtyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms4-coumaric acid using tyrosine as a substrate; (ii) nucleic acidsequence encoding phenylalanine ammonia lyase, wherein the encodedphenylalanine ammonia lyase converts phenylalanine to trans-cinnamicacid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase,wherein the cinnamate-4-hydroxylase produces 4-coumaric acid fromtrans-cinnamic acid; (iv) nucleic acid sequence encodingflavanone-3-hydroxylase, wherein flavanone-3-hydroxylase formsdihydrokaempferol from naringenin; and (v) any combinations thereof.

As shown in FIG. 4, in certain embodiments, the engineered host cellcomprises at least one or more nucleic acid sequences selected from: (i)nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii)nucleic acid sequences encoding phenylalanine ammonia lyase activity;(iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity;(iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL)activity; and (v) any combination thereof.

As shown in FIG. 4, in certain embodiments, the engineered host cellcomprises an exogenous nucleic acid sequence selected from the groupconsisting of: (i) nucleic acid sequence encoding tyrosine ammonialyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acidusing tyrosine as a substrate; (ii) nucleic acid sequence encodingphenylalanine ammonia lyase, wherein the encoded phenylalanine ammonialyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acidsequence encoding cinnamate-4-hydroxylase, wherein thecinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamicacid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligaseactivity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA fromcoumaric acid (v) nucleic acid sequence encoding chalcone synthaseactivity, wherein chalcone synthase forms naringenin chalcone frommalonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encodingchalcone isomerase activity, wherein chalcone isomerase forms naringeninfrom naringenin chalcone; (vii) nucleic acid sequence encodingflavanone-3-hydroxylase, wherein flavanone-3-hydroxylase formsdihydrokaempferol from naringenin; and (viii) any combinations thereof.

The compositions as described above, can be used in methods describedherein for increasing the production of flavonoids or anthocyanins. Suchmethods involve providing any of the compositions described above toresult in enzymatic transformation by the engineered host cell ofglycerol through multiple chemical intermediates into a flavonoid oranthocyanin (such as shown in part or in whole in FIG. 4).

In yet another aspect, it is envisioned that the pathway illustrated inFIG. 4 can be carried out using a plurality of engineered host cells, asopposed to a single host cell as described above. In such embodiments,the plurality of the engineered host cells have one or more geneticmodifications that result in enzymatic transformation by the engineeredhost cell of glycerol through multiple chemical intermediates into aflavonoid or anthocyanin (as shown in FIG. 4).

Aspects of the invention are now described with reference herein to FIG.4.

Step 1: conversion of pyruvate to acetate. poxB is deleted to reducecarbon loss and eliminate the byproducts.

Step 2: conversion of pyruvate to lactate. ldhA is deleted to reducecarbon loss and eliminate the byproducts.

Step 3: conversion of Acetyl-CoA to acetate. ackA-pta is deleted toreduce carbon loss and eliminate the byproducts.

Step 4: conversion of Acetyl-CoA to ethanol (EtOH). adhE is deleted toreduce carbon loss and eliminate the byproducts.

Step 5: conversion of acetyl-CoA to a substrate for the tricarboxylicacid cycle (TCA).

Step 6: conversion of acetyl-CoA to mal-CoA. Heterologous ACC isexpressed to increase the concentration of available mal-CoA.Heterologous ACC may be obtained from fungal species. Accordingly,embodiments of the invention provide an engineered host cell thatcomprises one or more genetic modifications to increase the productionand/or availability of malonyl-CoA. In certain embodiments, theengineered host cell comprises one or more genetic modificationsselected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii)overexpression of acetyl-CoA carboxylase. In another embodiment, theengineered host cell is an E. coli. In certain embodiments, theacetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorulatoruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs ofacetyl-CoA carboxylase having at least 50% amino acid identity to theacetyl-CoA carboxylase of these aforementioned species. In certainembodiments, one or more genetic modification is deletion or attenuationof one or more fatty biosynthetic genes resulting in decrease in fattyacid biosynthesis. In certain embodiments, one or more geneticmodification is overexpression of acetyl-CoA synthase (ACS). In certainembodiments, the acetyl-CoA synthase is selected from: acetyl-CoAsynthase gene of E. coli, acetyl-CoA synthase gene of Salmonellatyphimurium, and orthologs of acetyl-CoA synthase gene in any otherspecies having at least 50% amino acid identity to the acetyl-CoAsynthase gene of E. coli and Salmonella typhimurium. In certainembodiments, one or more genetic modification is selected from a groupconsisting of: (i) overexpression a gene encoding pyruvate dehydrogenase(PDH), wherein the PDH may include E354K mutation; (ii) exogenousnucleic acid sequence encoding a malonyl-CoA synthetase; (iii)upregulation of endogenous pantothenate kinase (PanK), wherein PanK isnot feedback inhibited by coenzyme A; (iv) exogenous nucleic acidsequence encoding a malonate transporter; and (v) any combinationsthereof. In certain embodiments, the malonyl-CoA synthetase is selectedfrom of malonyl-CoA synthetases of Streptomyces coelicolor,Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least50% identity to any of these or other naturally occurring malonyl-CoAsynthetases. In certain embodiments, one or more genetic modificationsto decrease fatty acid biosynthesis is selected from: (i) mutation ordownregulation of a gene encoding malonyl-CoA-ACP transacylase (E. colifabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E.coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme(E. coli fabB); (iv) downregulation of acyl carrier protein (E. coliacpP); and (v) any combinations thereof. In certain embodiments, theengineered host cell comprises peptides selected from: (i) acetyl-CoAcarboxylase (ACC) having an amino acid sequence at least 80% identicalto the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii)malonate CoA-transferase having an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO: 19; (iii)acetyl-CoA synthase (ACS) having an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO: 16; (iv)malonyl-CoA synthase having an amino acid sequence at least 80%identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonatetransporter having an amino acid sequence at least 80% identical to SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinasehaving an amino acid sequence at least 80% identical to SEQ ID NO: 88,SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.

In another aspect, the invention provides a method of increasing theproduction of flavonoids comprising an engineered host cell, wherein theone or more engineered host cells comprise one or more geneticmodifications to increase production and/or availability of malonyl-CoA.In certain embodiments, the engineered host cell comprises one or moregenetic modifications selected from: (i) expression of acetyl-CoAcarboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. Inanother embodiment, the engineered host cell is an E. coli. In certainembodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides,Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, andorthologs of acetyl-CoA carboxylase having at least 50% amino acididentity to the acetyl-CoA carboxylase of these aforementioned species.In certain embodiments, one or more genetic modification is deletion orattenuation of one or more fatty biosynthetic genes resulting indecrease in fatty acid biosynthesis. In certain embodiments, one or moregenetic modification is overexpression of acetyl-CoA synthase (ACS). Incertain embodiments, the acetyl-CoA synthase is selected from:acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene ofSalmonella typhimurium, and orthologs of acetyl-CoA synthase gene in anyother species having at least 50% amino acid identity to the acetyl-CoAsynthase gene of E. coli and Salmonella typhimurium. In certainembodiments, one or more genetic modification is selected from a groupconsisting of: (i) overexpression a gene encoding pyruvate dehydrogenase(PDH), wherein the PDH may include E354K mutation; (ii) exogenousnucleic acid sequence encoding a malonyl-CoA synthetase; (iii)upregulation of endogenous pantothenate kinase (PanK), wherein PanK isnot feedback inhibited by coenzyme A; (iv) exogenous nucleic acidsequence encoding a malonate transporter; and (v) any combinationsthereof. In certain embodiments, the malonyl-CoA synthetase is selectedfrom of malonyl-CoA synthetases of Streptomyces coelicolor,Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least50% identity to any of these or other naturally occurring malonyl-CoAsynthetases. In certain embodiments, one or more genetic modificationsto decrease fatty acid biosynthesis is selected from: (i) mutation ordownregulation of a gene encoding malonyl-CoA-ACP transacylase (E. colifabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E.coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme(E. coli fabB); (iv) downregulation of acyl carrier protein (E. coliacpP); and (v) any combinations thereof. In certain embodiments, theengineered host cell comprises peptides selected from: (i) acetyl-CoAcarboxylase (ACC) having an amino acid sequence at least 80% identicalto the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii)malonate CoA-transferase having an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO: 19; (iii)acetyl-CoA synthase (ACS) having an amino acid sequence at least 80%identical to the polypeptide set forth in SEQ ID NO: 16; (iv)malonyl-CoA synthase having an amino acid sequence at least 80%identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonatetransporter having an amino acid sequence at least 80% identical to SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84,SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinasehaving an amino acid sequence at least 80% identical to SEQ ID NO: 88,SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.

Step 7: conversion of mal-CoA to malonyl-ACP (acyl carrier protein).malonyl-coA-ACP transacylase (fabD) is downregulated to increase carbonflux.

Step 8: conversion of malonyl-ACP to 3-ketyoacyl-ACP. beta-ketoacyl-ACPsynthase II (fabF) is downregulated to increase carbon flux.

Step 9: conversion to mal-CoA to naringenin chalcone; conversion ofcoumaryl-CoA to naringenin chalcone. A heterologous CHS isoverexpressed.

Step 10: conversion to naringenin chalcone to naringenin. A heterologousCHI is overexpressed.

Steps 11, 12, and 13: conversion of naringenin to dihydrokaempferol(DHK); conversion of naringenin to eriodictyol (EDL); conversion oferiodictyol (EDL) to dihydroquercetin (DHQ); conversion of (DHK) todihydroquercetin (DHQ); conversion of dihydrokaempferol (DHK) todihydromyricetin (DHM); conversion of pentahydroxyflayaone (PHF) todihydromyricein (DHM). Heterologous F3′5′H, F3H, F3H, and/or CPR areoverexpressed. Accordingly, as shown in FIG. 4, in another aspect, theinvention provides method of increasing the production ofdihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/orpentahydroxyflayaone (PHF) comprising an engineered host cell, whereinthe engineered host cell comprises cytochrome P450 reductase (CPR) andat least one of flavanone-3′-hydroxylase (F3′H) or flavonoid3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor forincrease in production of dihydroquercetin (DHQ), dihydromyricein (DHM),eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/ordihydrokaempferol (DHK). In certain embodiments, the engineered hostcell further comprises peptides selected from a group consisting of: (i)flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR);and (iii) any combination thereof. In certain embodiments, theengineered host cell produces eriodictyol or taxifolin. In certainembodiments, the engineered host cell further comprises flavonoid3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered hostcell produces pentahydroxyflavone or dihydromyricetin. In certainembodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove theN-terminal leader sequence. In certain embodiments, cytochrome P450reductase (CPR) is truncated to remove the N-terminal leader sequence.In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused withcytochrome P450 reductase (CPR). In certain embodiments, flavonoid3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase(CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has anamino acid sequence at least 80% identical to the polypeptide set forthin SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H)has an amino acid sequence at least 80% identical to the polypeptide setforth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase(CPR) has an amino acid sequence at least 80% identical to thepolypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80%identical to the polypeptides selected from a group consisting of: (i)SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certainembodiments, the engineered host cell further comprises cytochrome b₅.In certain embodiments, cytochrome b₅ has an amino acid sequence atleast 80% identical to the polypeptide set forth in SEQ ID NO. 98.

As shown in FIG. 4, in another aspect, the invention provides method ofincreasing the production of dihydroquercetin (DHQ), dihydromyricein(DHM), eriodictyol (EDL), and/or pentahydroxyflayaone (PHF) comprisingan engineered host cell, wherein the engineered host cell comprisescytochrome P450 reductase (CPR) and at least one offlavanone-3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H).In certain embodiments, the precursor for increase in production ofdihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/orpentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK).In certain embodiments, the engineered host cell further comprisespeptides selected from a group consisting of: (i) flavonoid3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii)any combination thereof. In certain embodiments, the engineered hostcell produces eriodictyol or taxifolin. In certain embodiments, theengineered host cell further comprises flavonoid 3′,5′-hydroxylase(F3′5′H). In certain embodiments, the engineered host cell producespentahydroxyflavone or dihydromyricetin. In certain embodiments,flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminalleader sequence. In certain embodiments, cytochrome P450 reductase (CPR)is truncated to remove the N-terminal leader sequence. In certainembodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochromeP450 reductase (CPR). In certain embodiments, flavonoid3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase(CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has anamino acid sequence at least 80% identical to the polypeptide set forthin SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H)has an amino acid sequence at least 80% identical to the polypeptide setforth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase(CPR) has an amino acid sequence at least 80% identical to thepolypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80%identical to the polypeptides selected from a group consisting of: (i)SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certainembodiments, the engineered host cell further comprises cytochrome b₅.In certain embodiments, cytochrome b₅ has an amino acid sequence atleast 80% identical to the polypeptide set forth in SEQ ID NO. 98.

Step 14: conversion of dihydroquercetin (DHQ) to leucocyanidin (LC);conversion of dihydrokaempferol (DHK) to leucopelargonidin (LP); andconversion of dihydromyricetin (DHM) to leucodelphinidin (LD).Heterologous DFR is overexpressed.

Step 15: conversion of leucocyanidin (LC) to catechin; conversion ofleucodelphinidin (LD) to gallocatechin; and conversion ofleucopelargonidin (LP) to afzelechin. Heterologous LAR is overexpressed.

Step 16: conversion of catechin to cyanidin; conversion of leucocyanidin(LC) to catechin; conversion to leucodelphinidin (LD) to delphinidin;conversion of gallocatechin to delphinidin; conversion ofleucopelargonidin (LP) to pelargonidin; or conversion of afzelechin topelargonidin. Heterologous ANS is overexpressed. Step 16 could becarried in vivo or in a cell-free medium. Accordingly, as shown in FIG.4, in another aspect, the invention provides an engineered host cell,wherein the engineered host cell comprises one or more geneticmodifications to increase transformation of leucocyanidin or catechin tocyanidin-3-glucoside (Cy3G). In certain embodiments, one or more geneticmodifications comprises overexpression of anthocyanin synthase. Incertain embodiments, the anthocyanin synthase is selected from: (i)anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) theanthocyanin synthase has an amino acid sequence at least 80% identicalto SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69;(iii) the anthocyanin synthase has an amino acid sequence at least 80%identical to SEQ. ID NO: 13; and (iv) any combinations thereof. Incertain embodiments, one or more engineered host cells comprisesflavonoid-3-glucosyl transferase (3GT). In certain embodiments,flavonoid-3-glucosyl transferase is selected from: (i)flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii)the flavonoid-3-glucosyl transferase has an amino acid sequence at least80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ.ID NO: 73; and (iii) any combinations thereof. In certain embodiments,one or more genetic modifications comprises overexpression ofanthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). Incertain embodiments, one or more genetic modifications comprisesoverexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more geneticmodifications comprises overexpression of anthocyanin synthase andflavonoid-3-glucosyl transferase (3GT). In certain embodiments, the oneor more genetic modifications are selected from a group consisting of:(i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT),and (iii) a combination thereof.

In another aspect, the invention provides a method for increasing theproduction of flavonoids comprising an engineered host cell, wherein theengineered host cell comprises one or more genetic modifications toincrease transformation of leucocyanidin or catechin tocyanidin-3-glucoside (Cy3G). In certain embodiments, one or more geneticmodifications comprises overexpression of anthocyanin synthase. Incertain embodiments, the anthocyanin synthase is selected from: (i)anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) theanthocyanin synthase has an amino acid sequence at least 80% identicalto SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69;(iii) the anthocyanin synthase has an amino acid sequence at least 80%identical to SEQ. ID NO: 13; and (iv) any combinations thereof. Incertain embodiments, one or more engineered host cells comprisesflavonoid-3-glucosyl transferase (3GT). In certain embodiments,flavonoid-3-glucosyl transferase is selected from: (i)flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii)the flavonoid-3-glucosyl transferase has an amino acid sequence at least80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ.ID NO: 73; and (iii) any combinations thereof. In certain embodiments,one or more genetic modifications comprises overexpression ofanthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). Incertain embodiments, one or more genetic modifications comprisesoverexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more geneticmodifications comprises overexpression of anthocyanin synthase andflavonoid-3-glucosyl transferase (3GT). In certain embodiments, the oneor more genetic modifications are selected from a group consisting of:(i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT),and (iii) a combination thereof.

In another aspect, the invention provides a method of increasing thetransformation of leucocyanidin or catechin to cyanidin-3-glucoside(Cy3G) comprising anthocyanin synthase, wherein the anthocyanin synthaseis selected from: (i) anthocyanin synthase of Carica papaya (SEQ. IDNO:13); (ii) the anthocyanin synthase has an amino acid sequence atleast 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68,or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acidsequence at least 80% identical to SEQ. ID NO: 13; and (iv) anycombinations thereof.

In another aspect, the invention provides a method of increasing thetransformation of leucocyanidin or catechin to cyanidin-3-glucoside(Cy3G) comprising flavonoid-3-glucosyl transferase (3GT), wherein theflavonoid-3-glucosyl transferase is selected from: (i)flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii)the flavonoid-3-glucosyl transferase has an amino acid sequence at least80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ.ID NO: 73; and (iii) any combinations thereof.

Step 17: conversion of pelargonidin to callistephin; conversion ofdelphinidin to myrtillin (De3G); conversion of cyanidin to Cy3G.Heterologous 3GT was overexpressed in E. coli. Step 17 could be carriedin vivo or as a cell-free reaction.

Step 18: conversion of pyruvate to phosphoenolpyruvate (PEP). ppsA isoverexpressed to upregulate tyrosine.

Step 19: conversion of fructose-6-phosphate (F6P) toerythrose-4-phosphate (E4P). tktA is overexpressed to upregulatetyrosine.

Step 20: conversion of phosphoenolpyruvate (PEP) todeoxy-d-arabino-heptulosonate-7-phosphate (DAHP). aroG variant isoverexpressed to upregulate tyrosine.

Step 21: conversion of deoxy-d-arabino-heptulosonate-7-phosphate (DAHP)to dehydroquinate (DHQ); conversion of erythrose-4-phosphate (E4P) todehydroquinate (DHQ).

Step 22: conversion of dehydroquinate (DHQ) to 3-dehydroshikimate (DHS).

Step 23: conversion of 3-dehydroshikimate (DHS) to shikimic acid (SHK).aroE is overexpressed to upregulate tyrosine.

Step 24: conversion of shikimic acid (SHK) to shikimate-3-phosphate(S3P).

Step 25: conversion of shikimate-3-phosphate (S3P) to5-enolpyruvylshikimate-3-phosphate (EPSP).

Step 26: conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) tochorismic acid (CHA).

Step 27: conversion of chorismic acid (CHA) to prephenate (PPA);conversion of prephenate (PPA) to 4-hydroxy-phenylpyruvate (HPP). tryAvariant is overexpressed.

Step 28: conversion of 4-hydroxy-phenylpyruvate (HPP) to tyrosine;conversion of phenylpyruvate (POPP) to phenylalanine (Phe). Accordingly,as shown in FIG. 4, embodiments of the invention provide an engineeredhost cell, wherein the engineered host cell comprises one or moregenetic modifications to increase endogenous biosynthesis of tyrosine.In certain embodiments, one or more genetic modifications comprisesupregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certainembodiments, one or more genetic modifications are selected from: (i)upregulation of chorismate mutase; (ii) upregulation of prephenatedehydrogenase; (iii) overexpression of shikimate kinase; (iv)overexpression of shikimate dehydrogenase; and (v) any combinationsthereof. In certain embodiments, one or more genetic modificationscomprises downregulation of L-phenylalanine biosynthetic pathway. Incertain embodiments, one or more genetic modifications comprisesexpression of exogenous phosphoenolpyruvate synthase (ppsA). In certainembodiments, one or more genetic modifications comprises expression ofexogenous transketolase (tktA). In certain embodiments, wherein the oneor more genetic modifications comprises disruption of tyrR gene.

As shown in FIG. 4, in another aspect, the invention provides a methodof increasing endogenous biosynthesis of tyrosine comprising anengineered cell, wherein the engineered host cell comprises one or moregenetic modifications to increase endogenous biosynthesis of tyrosine.In certain embodiments, one or more genetic modifications comprisesupregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certainembodiments, one or more genetic modifications are selected from: (i)upregulation of chorismate mutase; (ii) upregulation of prephenatedehydrogenase; (iii) overexpression of shikimate kinase; (iv)overexpression of shikimate dehydrogenase; and (v) any combinationsthereof. In certain embodiments, one or more genetic modificationscomprises downregulation of L-phenylalanine biosynthetic pathway. Incertain embodiments, one or more genetic modifications comprisesexpression of exogenous phosphoenolpyruvate synthase (ppsA). In certainembodiments, one or more genetic modifications comprises expression ofexogenous transketolase (tktA). In certain embodiments, wherein the oneor more genetic modifications comprises disruption of tyrR gene.

Step 29: conversion of tyrosine to coumaric acid. A heterologous TAL isoverexpressed.

Step 30: conversion of courmaric acid to coumaryl-CoA. A heterologous4CL is overexpressed.

Step 31: conversion of glutamate (Glut) to glutamyl-tRNA.

Step 32: conversion of glutamyl-tRNA to glutamate semialdehyde (GSA).hemA is overexpressed to upregulate ALA.

Step 33: conversion of glutamate semialdehyde (GSA) to δ amino levulinicacid (ALA). hemL is overexpressed to upregulate ALA.

Step 34: conversion of δ amino levulinic acid (ALA) to porphobilinogen(PBG).

Step 35: conversion of porphobilinogen (PBG) to hydroxymethylbilane(HMB).

Step 36: conversion of hydroxymethylbilane (HMB) to uroporphyrinogen III(UPPIII).

Step 37: conversion of uroporphyrinogen III (UPPIII) tocoproporphyrinogen III (CPPIII).

Step 38: conversion of coproporphyrinogen III (CPPIII) toprotoporphyrinogen IX (PPPIX).

Step 39: conversion of protoporphyrinogen IX (PPPIX) to protoporphyrinIX, which is subsequently covered to heme.

Step 40: conversion of prephenate (PPA) to phenylpyruvate (POPP).

Step 41: conversion of phenylalanine (Phe) to cinnamate. HeterologousPAL and/or TAL are overexpressed.

Step 42: conversion of cinnamate to coumaric acid. Heterologous C4H/CPRare overexpressed.

TABLE 11 Enzyme Sequences: Enzyme: Sequence: SEQ ID: Tyrosine ammonia-MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTDEEIVRMGAS  1 lyase (TAL)ARTIEEYLKSDKPIYGLTQGFGPLVLFDADSELEQGGSLISHLGT SaccharothrixGQGAPLAPEVSRLILWLRIQNMRKGYSAVSPVFWQKLADLWNKGF espanaensisTPAIPRHGTVSASGDLQPLAHAALAFTGVGEAWTRDADGRWSTVP Accession:AVDALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHRSALRLV ABC88669.1RACAVLSARLATLLGANPEHYDVGHGVARGQVGQLTAAEWIRQGLPRGMVRDGSRPLQEPYSLRCAPQVLGAVLDQLDGAGDVLAREVDGCQDNPITYEGELLHGGNFHAMPVGFASDQIGLAMHMAAYLAERQLGLLVSPVTNGDLPPMLTPRAGRGAGLAGVQISATSFVSRIRQLVFPASLTTLPTNGWNQDHVPMALNGANSVFEALELGWLTVGSLAVGVAQLAAMTGHAAEGVWAELAGICPPLDADRPLGAEVRAARDLLSAH ADQLLVDEADGKDFGPhenylalanine MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAKGTFEAFTFH  2ammonia-lyase ISEEANKRIEECNELKHEIMNQHNPIYGVTTGFGDSVHRQISGEK (PAL)AWDLQRNLIRFLSCGVGPVADEAVARATMLIRTNCLVKGNSAVRL BrevibacillusEVIHQLIAYMERGITPIIPERGSVGASGDLVPLSYLASILVGEGK laterosporus LMGVLYKGEEREVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFACL 15441AYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQKPHLGQMA Accession:SAKNIYTLLEGSQLSKEYSQIVGNNEKLDSKAYLELTQSIQDRYS WP_003337219.1IRCAPHVTGVLYDTLDWVKKWLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQAMDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIPRFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVSVFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIHLIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERDRALDGDIEKVVQLIRSGNLK KEIHDQNVND Cinnamate-4-MDLLLIEKTLLALFAAIIGAIVISKLRGKRFKLPPGPLPVPIFGN  3 hydroxylase (C4H)WLQVGDDLNHRNLTDLAKKFGEIFLLRMGQRNLVVVSSPDLAKEV Helianthus annuusLHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV L.PFFTNKVVQQYRYGWEAEAAAVVEDVKKNPAAATEGVVIRRRLQL Accession:MMYNNMFRIMFDRRFESEDDPLFVKLKALNGERSRLAQSFEYNYG QJC72299.1DFIPILRPFLKGYLKLCKEVKEKRFQLFKDYFVDERKKLESTKSVDNNQLKCAIDHILDAKEKGEINEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQAKLRNELDTKLGPGVQVTEPDLHKLPYLQAVIKETLRLRMAIPLLVPHMNLHDAKLGGYDIPAESKILVNAWWLANNPEQWKKPEEFRPERFFEEESKVEANGNDFRYLPFGVGRRSCPGIILALPILGITIGRLVQNFELLPPPGQSKVDTTEKGGQFSLHILKH STIVAKPRAL 4-coumarate-CoAMGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKVGDKSCL  4 ligase (4CL)INGATGETFTYSQVELLSRKVASGLNKLGIQQGDTIMLLLPNSPE PetroselinumYFFAFLGASYRGAISTMANPFFTSAEVIKQLKASQAKLIITQACY crispumVDKVKDYAAEKNIQIICIDDAPQDCLHFSKLMEADESEMPEVVIN Accession:SDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYMH P14912.1SEDVMICILPLFHIYSLNAVLCCGLRAGVTILIMQKFDIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDKYDLSSVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIVDPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRTTIDEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRTNGFTTTEEEIKQFVSKQVVFYKRIFRVFFVDAIPKSPSGKILRKDLRARIASG DLPK Chaicone synthaseMVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTYPDYYFRITN  5 (CHS)SEHKTDLKEKFKRMCEKSMIKKRYMHLTEEILKENPSMCEYMAPS Petunia x hybridaLDARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGV Accession:DMPGCDYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN AAF60297.1NKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAGAIIIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLEEAFRPLSISDWNSLFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNYGNMSSACVLFILDEMRKASAKEGLGTTGEGLEWGVLFGFGPGLTVETVVLHSVAT Chalcone isomeraseMAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERGLTIEGNFI  6 (CHI)KFTAIGVYLEDIAVASLAAKWKGKSSEELLETLDFYRDIISGPFE Medicago sativaKLIRGSKIRELSGPEYSRKVMENCVAHLKSVGTYGDAEAEAMQKF Accession:AEAFKPVNFPPGASVFYRQSPDGILGLSFSPDTSIPEKEAALIEN P28012.1KAVSSAVLETMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN Flavanone 3-MAPTPTTLTAIAGEKTLQQSFVRDEDERPKVAYNQFSNEIPIISL  7 hydroxylase (F3H)SGIDEVEGRRAEICNKIVEACEDWGVFQIVDHGVDAKLISEMTRL Rubus occidentalisARDFFALPPEEKLRFDMSGGKKGGFIVSSHLQGEAVQDWREIVTY Accession:FSYPVRHRDYSRWPDKPEGWRAVTQQYSDELMGLACKLLEVLSEA ACM17897.1MGLEKEALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHFLSNGRFKNADHQAVVNSNHSRLSIATFQNPAQEAIVYPLKVREGEKPILEEPITYTEMYKKKMSKDLELARLKKLAKEQQPEDSEKAKLEVKQV DDIFA Flavonoid 3′MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIGNLPHM  8 hydroxylase (F3′H)GPKPHQTLAAMVTTYGPILHLRLGFADVVVAASKSVAEQFLKVHD Brassica napusANFASRPPNSGAKHMAYNYQDLVFAPYGQRWRMLRKISSVHLFSA Accession:KALEDFKHVRQEEVGTLMRELARANTKPVNLGQLVNMCVLNALGR ABC58723.1EMIGRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFVPALDCLDLQGVAGKMKRLHKRFDAFLSSILEEHEAMKNGQDQKHTDMLSTLISLKGTDFDGEGGTLTDTEIKALLLNMFTAGTDTSASTVDWAIAELIRHPEIMRKAQEELDSVVGRGRPINESDLSQLPYLQAVIKENFRLHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDPLTFRPERFLPGGEKAGVDVKGNDFELIPFGAGRRICAGLSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNMEETYGITLQRAVPLVV HPKLRLDMSAYGLGSACytochrome P450 MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPAVMAMLLE  9reductase (CPR) NKELVMILTTSVAVLIGCVVVLIWRRSSGSGKKVVEPPKLIVPKSCatharanthus VVEPEEIDEGKKKFTIFFGTQTGTAEGFAKALAEEAKARYEKAVI roseusKVIDIDDYAADDEEYEEKFRKETLAFFILATYGDGEPTDNAARFY Accession:KWFVEGNDRGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVAEQ Q05001GGKRIVPLVLGDDDQCIEDDFAAWRENVWPELDNLLRDEDDTTVSTTYTAAIPEYRVVFPDKSDSLISEANGHANGYANGNTVYDAQHPCRSNVAVRKELHTPASDRSCTHLDFDIAGTGLSYGTGDHVGVYCDNLSETVEEAERLLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCTLRTALTRYADLLNTPKKSALLALAAYASDPNEADRLKYLASPAGKDEYAQSLVANQRSLLEVMAEFPSAKPPLGVFFAAIAPRLQPRFYSISSSPRMAPSRIHVTCALVYEKTPGGRIHKGVCSTWMKNAIPLEESRDCSWAPIFVRQSNFKLPADPKVPVIMIGPGTGLAPFRGFLQERLALKEEGAELGTAVFFFGCRNRKMDYIYEDELNHFLEIGALSELLVAFSREGPTKQYVQHKMAEKASDIWRMISDGAYVYVCGDAKGMARDVHRTLHTIAQEQGSMDSTQAEGFVKNLQMTGRYLRDVW Flavonoid 3′, 5′-MSTSLLLAAAAILFFITHLFLRFLLSPRRTRKLPPGPKGWPVVGA 10 hydroxylaseLPMLGNMPHAALADLSRRYGPIVYLKLGSRGMVVASTPDSARAFL (F3′5′H)KTQDLNFSNRPTDAGATHIAYNSQDMVFADYGPRWKLLRKLSSLH DelphiniumMLGGKAVEDWAVVRRDEVGYMVKAIYESSCAGEAVHVPDMLVFAM grandiflorumANMLGQVILSRRVFVTKGVESNEFKEMVIELMTSAGLFNVGDFIP Accession:SIAWMDLQGIVRGMKRLHKKFDALLDKILREHTATRRERKEKPDL BAO66642VDVLMDNRDNKSEQERLTDTNIKALLLNLFSAGTDTSSSTIEWALTEMIKNPSIFGRAHAEMDQVIGRNRRLEESDIPKLPYLQAICKETFRKHPSTPLNLPRVAIEPCEVEGYHIPKGTRLSVNIWAIGRDPNVWENPLEFNPDRFLTGKMAKIDPRGNNFELIPFGAGRRICAGTRMGIVLVEYILGSLVHAFEWKLRDGETLNMEETFGIALQKAVPLAAVV TPRLPPSAYVVDihydroflavonol 4- MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATVRDPSNMKKVK 11reductase (DFR) HLLDLPGAANRLTLWKADLVDEGSFDEPIQGCTGVFHVATPMDFE AnthuriumSKDPESEMIKPTIEGMLNVLRSCARASSTVRRVVFTSSAGTVSIH andraeanumEGRRHLYDETSWSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK Accession:NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPHYSILNP AAP20866.1VQFVHLDDLCNAHIFLFECPDAKGRYICSSHDVTIAGLAQILRQRYPEFDVPTEFGEMEVFDIISYSSKKLTDLGFEFKYSLEDMFDGAIQSCREKGLLPPATKEPSYATEQLIATGQDNGH LeucoanthocyanidinMTVSGAIPSMTKNRTLVVGGTGFIGQFITKASLGFGYPTFLLVRP 12 reductase (LAR)GPVSPSKAVIIKTFQDKGAKVIYGVINDKECMEKILKEYEIDVVI DesmodiumSLVGGARLLDQLTLLEAIKSVKTIKRFLPSEFGHDVDRTDPVEPG uncinatumLTMYKEKRLVRRAVEEYGIPFTNICCNSIASWPYYDNCHPSQVPP Accession:PMDQFQIYGDGNTKAYFIDGNDIGKFTMKTIDDIRTLNKNVHFRP Q84V83.1SSNCYSINELASLWEKKIGRTLPRFTVTADKLLAHAAENIIPESIVSSFTHDIFINGCQVNFSIDEHSDVEIDTLYPDEKFRSLDDCYEDFVPMVHDKIHAGKSGEIKIKDGKPLVQTGTIEEINKDIKTLVETQ PNEEIKKDMKALVEAVPISAMGAnthocyanin MFSSVAVPRVEILASSGIESIPKEYVRPQEELTTIGNIFDEEKKD 13dioxygenase (ANS) EGPQVPTIDLRDIDSDDQQVRQRCRDELKKAAVDWGVMHLVNHGICarica papaya PDHLIDRVKKAGQAFFELPVEVKEKYANDQASGNIQGYGSKLANN Accession:ASGQLEWEDYYFHLIFPEEKRDLAIWPNNPADYIEVTSEYARQLR XP_021901846.1RLVSKILGVLSLGLGLEEGRLEKEVGGLDELLLQMKINYYPTCPQPELALGVEAHTDISALTFILHNMVPGLQLFYEGKWVTAKCVPNSIVMHVGDTIEILSNGKYKSILHRGLVNKEKVRISWAVFCEPPKEKIILKPLPETVSENEPPLFPPRTFAQHIQHKLFRKNQENLEAK Anthocy anidin-3-MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAVAAPHAVFSFFST 14 O-glycotransferaseSESNASIFHDSMHTMQCNIKSYDVSDGVPEGYVFTGRPQEGIDLF (3GT)MRAAPESFRQGMVMAVAETGRPVSCLVADAFIWFAADMAAEMGVA Vitis labruscaWLPFWTAGPNSLSTHVYIDEIREKIGVSGIQGREDELLNFIPGMS Accession:KVRFRDLQEGIVFGNLNSLFSRLLHRMGQVLPKATAVFINSFEEL ABR24135DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQWLKERKPTSVVYISFGTVTTPPPAELVALAEALEASRVPFIWSLRDKARMHLPEGFLEKTRGHGMVVPWAPQAEVLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFFGDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQILSQEKGKKLRENLRALRETADRAVGPKGSSTENFKTLVDLV SKPKDV Acetyl-CoAMVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHEGHTVISKVLIAN 15 carboxylase (ACC)NGMAAMKEIRSVRKWAYETFGNERAIEFTVMATPEDLKANAEYIR MucorMADNFVEVPGGSNNNNYANVELIVDVAERTAVHAVWAGWGHASEN circinelloidesPRLPEMLAKSKHKCLFIGPPASAMRSLGDKISSTIVAQSAQVPTM 1006PhLGWSGDGITETEFDAAGHVIVPDNAYNEACVKTAEQGLKAAEKIGF Accession:PVMIKASEGGGGKGIRMVKDGSNFAQLFAQVQGEIPGSPIFIMKL EPB82652.1AGNARHLEVQLLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAKPDVFEQMEKAAVRLGKLVGYVSAGTVEYLYSHHDDQFYFLELNPRLQVEHPTTEMVSGVNLPAAQLQIAMGIPLHRIRDIRVLYGVQPNSASEIDFGFEHPTSLTSHRRPTPKGHVIACRITAENPDAGFKPSSGIMQELNFRSSTNVWGYFSVVSAGGLHEYADSQFGHIFAYGENRQQARKNMVIALKELSIRADFRSTVEYIIRLLETPDFEENTINTGWLDMLISKKLTAERPDTMLAVFCGAVTKAHMASLDCFQQYKQSLEKGQVPSKGSLKTVFTVDFIYEEVRYNFTVTQSAPGIYTLYLNGTKTQVGIRDLSDGGLLISIDGKSHTTYSRDEVQATRMMVDGKTCLLEKESDPTQLRSPSPGKLVNLLVENGDHLNAGDAYAEIEVMKMYMPLIATEDGHVQFIKQAGATLEAGDIIGILSLDDPSRVKHALPFNGTVPAFGAPHITGDKPVQRFNATKLTLQHILQGYDNQALVQTVVKDFADILNNPDLPYSELNSVLSALSGRIPQRLEASIHKLADESKAANQEFPAAQFEKLVEDFAREHITLQSEATAYKNSVAPLSSIFARYRNGLTEHAYSNYVELMEAYYDVEILFNQQREEEVILSLRDQHKDDLDKVLAVTLSHAKVNIKNNVILMLLDLINPVSTGSALDKYFTPILKRLSEIESRATQKVTLKARELLILCQLPSYEERQAQMYQILKNSVTESVYGGGSEYRTPSYDAFKDLIDTKFNVFDVLPHFFYHADPYIALAAIEVYCRRSYHAYKILDVAYNLEHKPYVVAWKFLLQTAANGIDSNKRIASYSDLTFLLNKTEEEPIRTGAMTACNSLADLQAELPRILTAFEEEPLPPMLQRNAAPKEERMENILNIAVRADEDMDDTAFRTKICEMITANADVFRQAHLRRLSVVVCRDNQWPDYYTFRERENYQEDETIRHIEPAMAYQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDCRFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLEIVSHEYKNSDCNHLFINFIPTFAIEADDVEHALKDFVDRHGKRLWKLRVTGAEIRFNVQSKKPDAPIIPMRFTVDNVSGFILKVEVYQEVKTEKSGWILKSVNKIPGAMHMQPLSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQSVQNQWTQAIKRNPLLKQPSHLVEAKELVLDEDDVLQEIDRAPGTNTVGMVAWIMTIRTPEYPSGRRIIAIANDITFKIGSFGVAEDQVFYKASELARALGIPRIYLSANSGARIGLADELISQFRAAWKDASNPTAGFKYLYLTPAEYDVLAQQGDAKSVLVEEIQDEGETRLRITDVIGHTDGLGVENLKGSGLIAGATSRAYDDIFTITLVTCRSVGIGAYLVRLGQRTIQNEGQPIILTGAPALNKVLGREVYTSNLQLGGTQIMYKNGVSHLTAENDLEGIAKIVQWLSFVPDVRNAPVSMRLGADPIDRDIEYTPPKGPSDPRFFLAGKSENGKWLSGFFDQDSFVETLSGWARTVVVGRARLGGIPMGVVSVETRTVENIVPADPANSDSTEQVFMEAGGVWFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGGQRDMYNEVLKYGAQIVDALSNYKQPVFVYIIPNGELRGGAWVVVDPTINKDMMEMYADNNARGGVLEPEGIVEIKYRKPALLATMERLDATYASLKKQLAEEGKTDEEKAALKVQVEAREQELLPVYQQISIQFADLHDRAGRMKAKGVIRKALDWRRARHYFYWRVRRRLCEEYTFRKIVTATSAAPMPREQMLDLVKQWFTNDNETVNFEDADELVSEWFEKRASVIDQRISKLKSDATKEQIVSLGNADQEAVIEGFSQLIENLSEDAR AEILRKLNSRF Acetyl-CoAMSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTFWGEQGKI 16 synthase (ACS)LDWITPYQKVKNTSFAPGNVSIKWYEDGTLNLAANCLDRHLQENG SalmonellaDRTAIIWEGDDTSQSKHISYRELHRDVCRFANTLLDLGIKKGDVV typhimuriumAIYMPMVPEAAVAMLACARIGAVHSVIFGGFSPEAVAGRIIDSSS Accession:RLVITADEGVRAGRSIPLKKNVDDALKNPNVTSVEHVIVLKRTGS NP_463140.1DIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPLFILYTSGSTGKPKGVLHTTGGYLVYAATTFKYVFDYHPGDIYWCTADVGWVTGHSYLLYGPLACGATTLMFEGVPNWPTPARMCQVVDKHQVNILYTAPTAIRALMAEGDKAIEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKEKCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFFGVQPALVDNEGHPQEGATEGNLVITDSWPGQARTLFGDHERFEQTYFSTFKNMYFSGDGARRDEDGYYWITGRVDDVLNVSGHRLGTAEIESALVAHPKIAEAAVVGIPHAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRKEIGPLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNLGDTS TLADPGVVEKLLEEKQAIAMPSMalonyl-CoA MSSLFPALSPAPTGAPADRPALRFGERSLTYAELAAAAGATAGRI 17synthase (matB) GGAGRVAVWATPAMETGVAVVAALLAGVAAVPLNPKSGDKELAHIStreptomyces LSDSAPSLVLAPPDAELPPALGALERVDVDVRARGAVPEDGADDG coelicolorDPALVVYTSGTTGPPKGAVIPRRALATTLDALADAWQWTGEDVLV Accession:QGLPLFHVHGLVLGILGPLRRGGSVRHLGRFSTEGAARELNDGAT WP_011028356MLFGVPTMYHRIAETLPADPELAKALAGARLLVSGSAALPVHDHERIAAATGRRVIERYGMTETLMNTSVRADGEPRAGTVGVPLPGVELRLVEEDGTPIAALDGESVGEIQVRGPNLFTEYLNRPDATAAAFTEDGFFRTGDMAVRDPDGYVRIVGRKATDLIKSGGYKIGAGEIENALLEHPEVREAAVTGEPDPDLGERIVAWIVPADPAAPPALGTLADHVAARLAPHKRPRVVRYLDAVPRNDMGKIMKRALNRD MalonateMSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGTLVADLDA 18 transporter (matC)DGIFAGFPGDLFVVLVGVTYLFAIARANGTTDWLVHAAVRLVRGR StreptomycesVALIPWVMFALTGALTAIGAVSPAAVAIVAPVALSFATRYSISPL coelicolorLMGTMVVHGAQAGGFSPISIYGSIVNGIVEREKLPGSEIGLFLAS Accession:LVANLLIAAVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGSG NP_626686.1SDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGGAEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLTAVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTYVGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIVSAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAVSATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVYGGIVVAA VPALAWLVLVVPGFGMalonate CoA- MVKKRLWDKQRTRRQEKLNLAQQKGFAKQVEHARAIELLETVIAS 19transferase (MdcA) GDRVCLEGNNQKQADFLSKCLSQCNPDAVNDLHIVQSVLALPSHIAcinetobacter DVFEKGIASKVDFSFAGPQSLRLAQLVQQQKISIGSIHTYLELYGcalcoaceticus RYFIDLTPNICLITAHAADREGNLYTGPNTEDTPAIVEATAFKSG Accession:IVIAQVNEIVDKLPRVDVPADWVDFYIESPKHNYIEPLFTRDPAQ AAB97627.1ITEVQILMAMMVIKGIYAPYQVQRLNHGIGFDTAAIELLLPTYAASLGLKGQICTNWALNPHPTLIPAIESGFVDSVHSFGSEVGMEDYIKERPDVFFTGSDGSMRSNRAFSQTAGLYACDSFIGSTLQIELQGNSSTATVDRISGFGGAPNMGSDPHGRRHASYAYTKAGREATDGKLIKGRKLVVQTVETYREHMHPVFVEELDAWQLQDKMDSELPPIMIYGEDVTHIVTEEGIANLLLCRTDEEREQAIRGVAGYTPVGLKRDAAKVEELRQRGIIQRPEDLGIDPTQVSRDLLAAKSVKDLVKWSGGLYS PPSRFRNWPantothenate kinase MILELDCGNSLIKWRVIEGAARSVAGGLAESDDALVEQLTSQQAL 20(CoaX) PVRACRLVSVRSEQETSQLVARLEQLFPVSALVASSGKQLAGVRN PseudomonasGYLDYQRLGLDRWLALVAAHHLAKKACLVIDLGTAVTSDLVAADG aeruginosaVHLGGYICPGMTLMRSQLRTHTRRIRYDDAEARRALASLQPGQAT Accession:AEAVERGCLLMLRGFVREQYAMACELLGPDCEIFLTGGDAELVRD Q9HWCL1ELAGARIMPDLVFVGLALACPIE glutamyl-tRNAMTKKLLALGINHKTAPVSLRERVTFSPDTLDQALDSLLAQPMVQG 21 reductase (hemAm)GVVLSTCNRTELYLSVEEQDNLQEALIRWLCDYHNLNEDDLRNSL SalmonellaYWHQDNDAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQKGHL typhimuriumNASALRRMFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFE Accession:SLSTVTVLLVGAGETIELVARHLREHKVQKMIIANRTRERAQALA AAA88610.1DEVGAEVISLSDIDARLQDADIIISSTASPLPIIGKGMVERALKSRRNQPMLLVDIAVPRDVEPEVGKLANAYLYSVDDLQSIISHNLAQRQAAAVEAETIVEQEASEFMAWLRAQGASETIREYRSQSEQIRDELTTKALSALQQGGDAQAILQDLAWKLTNRLIHAPTKSLQQAARDG DDERLNILRDSLGLE5-aminolevulinic MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQWNRPDGGK 22acid synthase QDITVWCGNDYLGMGQHPVVLAAMHEALEAVGAGSGGTRNISGTT (ALAS)AYHRRLEAEIADLHGKEAALVFSSAYIANDATLSTLRLLFPGLII RhodobacterYSDSLNHASMIEGIKRNAGPKRIFRHNDVAHLRELIAADDPAAPK capsulatusLIAFESVYSMDGDFGPIKEICDIADEFGALTYIDEVHAVGMYGPR Accession:GAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIAASAKMVDAVRSY CAA37857APGFIFSTSLPPAIAAGAQASIAFLKTAEGQKLRDAQQMHAKVLKMRLKALGMPIIDHGSHIVPVVIGDPVHTKAVSDMLLSDYGVYVQPINFPTVPRGTERLRFTPSPVHDLKQIDGLVHAMDLLWARCA Tyrosine ammonia-MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTPALRERCAR 23 lyase (TAL)AHARLEHAIAEQRHIYGITTGFGPLANRLIGADQGAELQQNLIYH RhodobacterLATGVGPKLSWAEARALMLARLNSILQGASGASPETIDRIVAVLN capsulatus SB 1003AGFAPEVPAQGTVGASGDLTPLAHMVLALQGRGRMIDPSGRVQEA Accession:GAVMDRLCGGPLTLAARDGLALVNGTSAMTAIAALTGVEAARAID ADE84832.1AALRHSAVLMEVLSGHAEAWHPAFAELRPHPGQLRATERLAQALDGAGRVCRTLTAARRLTAADLRPEDHPAQDAYSLRVVPQLVGAVWDTLDWHDRVVTCELNSVTDNPIFPEGCAVPALHGGNFMGVHVALASDALNAALVTLAGLVERQIARLTDEKLNKGLPAFLHGGQAGLQSGFMGAQVTATALLAEMRANATPVSVQSLSTNGANQDVVSMGTIAARRARAQLLPLSQIQAILALALAQAMDLLDDPEGQAGWSLTARDLRDRIRAVSPGLRADRPLAGHIEAVAQGLRHPSAAADPPA Tyrosine ammonia-MITETNVAKPASTKVMNGDAAKAAPVEPFATYAHSQATKTVVIDG 24 lyase (TAL)HNMKVGDVVAVARHGAKVELAASVAGPVQASVDFKESKKHTSIYG TrichosporonVTTGFGGSADTRTSDTEALQISLLEHQLCGYLPTDPTYEGMLLAA cutaneumMPIPIVRGAMAVRVNSCVRGHSGVRLEVLQSFADFINIGLVPCVP Accession:LRGTISASGDLSPLSYIAGAICGHPDVKVFDTAASPPTVLTAPEA XP_018276715IAKYKLKTVRLASKEGLGLVNGTAVSAAAGALALYDAECLAMMSQTNTALTVEALDGHVGSFAPFIQEIRPHVGQIEAAKNIRHMLSNSKLAVHEEPELLADQDAGILRQDRYALRTSAQWIGPQLEMLGLARQQIETELNSTTDNPLIDVEGGMFHHGGNFQAMAVTSAMDSTRIVLQNLGKLSFAQVTELINCEMNHGLPSNLAGSEPSTNYHCKGLDIHCGAYCAELGFLANPMSNHVQSTEMHNQSVNSMAFASARKTMEANEVLSLLLGSQMYCATQALDLRVMEVKFKMAIVKLLNDTLTKHFSTFLTPEQLAKLNTTAAITLYKRLNQTPSWDSAPRFEDAAKHLVGCIMDALMVNDDITDLTNLPKWKKEFAKDAGDLYRSILTATTADGRNDLEPAEYLGQTRAVYEAIRSDLGVKVRRGDVAEGKSGKSIGSNVARIVEA MRDGRLMGAVSKMFFTyrosine ammonia- MNTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNFLKEFSG 25lyase (TAL) NKVIYGVNTGFGPMAQYRIKESDQIQLQYNLIRSHSSGTGKPLSP FlavobacteriumVCAKAAILARLNTLSLGNSGVHPSVINLMSELINKDITPLIFEHG johnsoniaeGVGASGDLVQLSHLALVLIGEGEVFYKGERRPTPEVFEIEGLKPI Accession:QVEIREGLALINGTSVMTGIGVVNVYHAKKLLDWSLKSSCAINEL WP_012023194VQAYDDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKREDHLYSGENTEEIFKEKVQEYYSLRCVPQILGPVLETINNVASILEDEFNSANDNPIIDVKNQHVYHGGNFHGDYISLEMDKLKIVITKLTMLAERQLNYLLNSKINELLPPFVNLGTLGFNFGMQGVQFTATSTTAESQMLSNPMYVHSIPNNNDNQDIVSMGTNSAVITSKVIENAFEVLAIEMITIVQAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYPF VQKVKDHLINNTyrosine ammonia- MSTTLILTGEGLGIDDVVRVARHQDRVELTTDPAILAQIEASCAY 26lyase (TAL) INQAVKEHQPVYGVTTGFGGMANVIISPEEAAELQNNAIWYHKTG HerpetosiphonAGKLLPFTDVRAAMLLRANSHMRGASGIRLEIIQRMVTFLNANVT aurantiacusPHVREFGSIGASGDLVPLISITGALLGTDQAFMVDFNGETLDCIS DSM 785ALERLGLPRLRLQPKEGLAMMNGTSVMTGIAANCVHDARILLALA Accession:LEAHALMIQGLQGTNQSFHPFIHRHKPHTGQVWAADHMLELLQGS ABX04526.1QLSRNELDGSHDYRDGDLIQDRYSLRCLPQFLGPIIDGMAFISHHLRVEINSANDNPLIDTASAASYHGGNFLGQYIGVGMDQLRYYMGLMAKHLDVQIALLVSPQFNNGLPASLVGNIQRKVNMGLKGLQLTANSIMPILTFLGNSLADRFPTHAEQFNQNINSQGFGSANLARQTIQTLQQYIAITLMFGVQAVDLRTHKLAGHYNAAELLSPLTAKIYHAVRSIVKHPPSPERPYIWNDDEQVLEAHISALAHDIANDGSLVSAVEQ TLSGLRSIILFR PhenylalanineMHDDNTSPYCIGQLGNGAVHGADPLNWAKTAKAMECSHLEEIKRM 27 ammonia-lyaseVDTYQNATQVMIEGATLTVPQVAAIARRPEVHVVLDAANARSRVD (PAL)ESSNWVLDRIMGGGDIYGVTTGFGATSHRRTQQGVELQRELIRFL PhyscomitrellaNAGVLSKGNSLPSETARAAMLVRTNTLMQGYSGIRWEILHAMEKL patensLNAHVTPKLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVTEDGR Accession:EVSALEALRIAGVEKPFELAPKEGLALVNGTAVGSALASTVCYDA XP_001758374.1NIMVLLAEVLSALFCEVMQGKPEFADPLTHKLKHHPGQMEAAAVMEWVLDGSSFMKAAAKFNETDPLRKPKQDRYALRTSPQWLGPQVEVIRNATHAIEREINSVNDNPIIDAARGIALHGGNFQGTPIGVSMDNMRLSLAAIAKLMFAQFSELVNDYYNNGLPSNLSGGPNPSLDYGMKGAEIAMASYLSEINYLANPVTTHVQSAEQHNQDVNSLGLVSARKTEEAMEILKLMSATFLVGLCQAIDLRHVEETMQSAVKQVVTQVAKKTLFMGSDGSLLPSRFCEKELLMVVDRQPVFSYIDDSTSDSYPLMEKLRGVLVSRALKSADKETSNAVFRQIPVFEAELKLQLSRVVPAVREAYDTKGLSLVPNRIQDCRTYPLYKLVRGDLKTQLLSGQRTVSPGQEIEKVFNAISAGQLVAPLLECVQGWTGTPGPFSARASC PhenylalanineMIETNHKDNFLIDGENKNLEINDIISISKGEKNIIFTNELLEFLQ 28 ammonia-lyaseKGRDQLENKLKENVAIYGINTGFGGNGDLIIPFDKLDYHQSNLLD (PAL)FLTCGTGDFFNDQYVRGIQFIIIIALSRGWSGVRPMVIQTLAKHL DictyosteliumNKGIIPQVPMHGSVGASGDLVPLSYIANVLCGKGMVKYNEKLMNA discoideum AX4SDALKITSIEPLVLKSKEGLALVNGTRVMSSVSCISINKFETIFK Accession:AAIGSIALAVEGLLASKDHYDMRIHNLKNHPGQILIAQILNKYFN XP_644510.1TSDNNTKSSNITFNQSENVQKLDKSVQEVYSLRCAPQILGIISENISNAKIVIKREILSVNDNPLIDPYYGDVLSGGNFMGNHIARIMDGIKLDISLVANHLHSLVALMMHSEFSKGLPNSLSPNPGIYQGYKGMQISQTSLVVWLRQEAAPACIHSLTTEQFNQDIVSLGLHSANGAASMLIKLCDIVSMTLIIAFQAISLRMKSIENFKLPNKVQKLYSSIIKIIPILENDRRTDIDVREITNAILQDKLDFFNLNL PhenylalanineMSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAKGTFEAFTFH 29 ammonia-lyaseISEEANKRIEECNELKHEIMNQHNPIYGVTTGFGDSVHRQISGEK (PAL)AWDLQRNLIRFLSCGVGPVADEAVARATMLIRTNCLVKGNSAVRL BrevibacillusEVIHQLIAYMERGITPIIPERGSVGASGDLVPLSYLASILVGEGK laterosporusVLYKGEEREVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFACL LMG 15441AYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQKPHLGQMA Accession:SAKNIYTLLEGSQLSKEYSQIVGNNEKLDSKAYLELTQSIQDRYS WP_003337219.1IRCAPHVTGVLYDTLDWVKKWLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQAMDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIPRFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVSVFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIHLIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERDRALDGDIEKVVQLIRSGNLK KEIHDQNVND Cinnamate-4-MDLLLMEKTLLGLFVAVVVAITVSKLRGKKFKLPPGPIPVPVFGN 30 hydroxylase (C4H)WLQVGDDLNHRNLTEMAKKFGEVFMLRMGQRNLVWSSPDLAKEVL Rubus sp. SSL-2007HTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVP Accession:FFTNKVVQQYRYGWESEAAAVVEDVKKHPEAATNGMVLRRRLQLM ABX74781.1MYNNMYRIMFDRRFESEDDPLFVKLKGLNGERSRLAQSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLFKDYFVDERKKLSSTQATTNEGLKCAIDHILDAQQKGEINEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQKKLRDELDTVLGRGVQITEPEIQKLPYLQAVVKETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPFGVGRRSCPGIILALPILGITLGRLVQNFELLPPPGQTQLDTTEKGGQFSLHILKHS PIVMKPRT Cinnamate-4-MDLLLLEKTLIGLFIAIVVAIIVSKLRGKKFKLPPGPIPVPVFGN 31 hydroxylase (C4H)WLQVGDDLNHRNLTDMAKKFGDVFMLRMGQRNLVVVSSPDLAKEV Fragaria vescaLHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV Accession:PFFTNKVVQQYRHGWEAEAAAVVEDVKKHPEAATSGMVLRRRLQL XP_004294725.1MMYNNMYRIMFDRRFESEEDPLFVKLKGLNGERSRLAQSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLFKDYFVDERKKLASTQVTTNEGLKCAIDHILDAQQKGEINEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQKKLRDELDTVLGHGVQVTEPELHKLPYLQAVVKETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPFGVGRRSCPGIILALPILGVTLGRLVQNFEMLPPPGQTQLDTTEKGGQFSLHILKH STIVMKPRA Cinnamate-4-MDLLLLEKTLIGLFFAILIAIIVSKLRSKRFKLPPGPIPVPVFGN 32 hydroxylase (C4H)WLQVGDDLNHRNLTEYAKKFGDVFLLRMGQRNLVVVSSPELAKEV Solanum tuberosumLHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV Accession:PFFTNKVVQQYRGGWESEAASVVEDVKKNPESATNGIVLRKRLQL ABC69046.1MMYNNMFRIMFDRRFESEDDPLFVKLRALNGERSRLAQSFEYNYGDFIPILRPFLRGYLKICKEVKEKRLKLFKDYFVDERKKLANTKSMDSNALKCAIDHILEAQQKGEINEDNVLYIVENFNVAAIETTLWSIEWGIAELVNHPHIQKKLRDEIDTVLGPGMQVTEPDMPKLPYLQAVIKETLRLRMAIPLLVPHMNLHDAKLAGYDIPAESKILVNAWWLANNPAHWKKPEEFRPERFFEEEKHVEANGNDFRFLPFGVGRRSCPGIILALPILGITLGRLVQNFEMLPPPGQSKLDTSEKGGQFSLHILKH STIVMKPRSF 4-coumarate-CoAMGDCAAPKQEIIFRSKLPDIYIPKHLPLHSYCFENISKVSDRACL 33 ligase (4CL)INGATGETFSYAQVELISRRVASGLNKLGIHQGDTMMILLPNTPE Daucus carotaYFFAFLGASYRGAVSTMANPFFTSPEVIKQLKASQAKLIITQACY Accession:VEKVKEYAAENNITVVCIDEAPRDCLHFTTLMEADEAEMPEVAID AIT52344.1SDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQRVDGENPNLYIHSEDVMICILPLFHIYSLNAVLCCGLRAGATILIMQKFDIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDNYDLSSVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIVDPETHASLPRNQSGEICIRGDQIMKGYLNDPESTKTTIDEEGWLHTGDIGFIDEDDELFIVDRLKEIIKYKGFQVAPAEIEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRLNGSTTTEEEIKQFVSKQVVFYKRVFRVFFVDAIPKSPSGKILRKELRARIASG DLPK 4-coumarate-CoAMEPTTKSKDIIFRSKLPDIYIPKHLPLHTYCFENISRFGSRPCLI 34 ligase (4CL)NGSTGEILTYDQVELASRRVGSGLHRLGIRQGDTIMLLLPNSPEF Striga asiaticaVLAFLGASHIGAVSTMANPFFTPAEVVKQAAASRAKLIVTQACHV Accession:DKVRDYAAEHGVKVVCVDGAPPEECLPFSEVASGDEAELPAVKIS GER48539.1PDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIHSDDVIMCVLPLFHIYSLNSIMLCGLRVGAAILIMQKFEIVPFLELIQRYRVTIGPFVPPIVLAIEKSPVVEKYDLSSVRTVMSGAAPLGRELEDAVRLKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVDTETGASLGRNQPGEICIRGDQIMKGYLNDPESTERTIDKEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEALLLNHPNISDAAVVSMKDEQAGEVPVAYVVKSNGSTITEDEIKQFVSKQVIFYKRINRVFFIDAIPKSPSGKILRKDLRARLAAG VPN 4-coumarate-CoAMPMENEAKQGDIIFRSKLPDIYIPNHLSLHSYCFENISEFSSRPC 35 ligase (4CL)LINGANNQIYTYADVELNSRKVAAGLHKQFGIQQKDTIMILLPNS Capsicum annuumPEFVFAFLGASYLGAISTMANPLFTPAEVVKQVKASNAEIIVTQA Accession:CHVNKVKDYALENDVKIVCIDSAPEGCVHFSELIQADEHDIPEVQ KAF3620179.1IKPDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIHSEDVMLCVLPLFHIYSLNSVLLCGLRVGAAILIMQKFDIVPFLELIQNYKVTIGPFVPPIVLAIAKSPMVDNYDLSSVRTVMSGAAPLGKELEDTVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVDPDTGNSLHRNQSGEICIRGDQIMKGYLNDPEATAGTIDKEGWLHTGDIGYIDNDDELFIVDRLKELIKYKGFQVAPAELEALLLNHPNISDAAVVPMKDEQAGEVPVAFVVRSNGSTITEDEVKEFISKQVIFYKRIKRVFFVDAVPKSPSGKILRKDLRAKLA AGFPN 4-coumarate-CoAMDTKTTQQEIIFRSKLPDIYIPKQLPLHSYCFENISQFSSKPCLI 36 ligase (4CL)NGSTGKVYTYSDVELTSRKVAAGFHNLGIQQRDTIMLLLPNCPEF Camellia sinensisVFAFLGASYLGAIITMANPFFTPAETIKQAKASNSKLIITQSSYT Accession:SKVLDYSSENNVKIICIDSPPDGCLHFSELIQSNETQLPEVEIDS ASU87409.1NEVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIHSEDMMMCVLPLFHIYSLNSVLLCGLRVGAAILIMQKFEIGSFLKLIQRYKVTIGPFVPPIVLAIAKSEVVDDYDLSTIRTMMSGAAPLGKELEDAVRAKFPHAKLGQGYGMTEAGPVLAMCLAFAKKPFEIKSGACGTVVRNAEMKIVDPDAGFSLPRNQPGEICIRGDQIMKGYLNDPEATERTIDKQGWLHTGDIGYIDDDDELFIVDRLKELIKYKGFQVAPAELEALLLNHPTISDAAVVPMKDESAGEVPVAFVVRTNGFEVTENEIKKYISEQVVFYKKINRVYFVDAIPKAPSGKILRKDLRARLAAGI PS Chaicone synthaseMVTVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTYPDYYFRITN 37 (CHS)SEHKTELKEKFKRMCEKSMIKTRYMHLTEEILKENPNMCAYMAPS Capsicum annuumLDARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGV Accession:DMPGCDYQLAKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN XP_016566084.1NKGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAAAIIMGSDPIPGVERPLFQLVSAAQTLLPDSEGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLVEAFQPLGISDWNSLFWIAHPGGPAILDQVELKLGLKPEKLKATREVLSNYGNMSSACVLFILDEMRKASTKEGLGTSGEGLEWGVLFGFGPGLTVETVVLHSVAI Chaicone synthaseMVTVEEVRKAQRAEGPATVLAIGTATPPNCIDQSTYPDYYFRITK 38 (CHS)SEHKAELKEKFQRMCDKSMIKKRYMYLTEEILKENPSMCEYMAPS Rosa chinensisLDARQDMVVVEIPKLGKEAATKAIKEWGQPKSKITHLVFCTTSGV Accession:DMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN AEC13058.1NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAIIVGSDPLPEVEKPLFELVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLNEAFKPLNITDWNSLFWIAHPGGPAILDQVEAKLGLKPEKLEATRHILSEYGNMSSACVLFILDEVRRKSAANGHKTTGEGLEWGVLFGFGPGLTVETVVLHSVAA Chaicone synthaseMSMTPSVHEIRKAQRSEGPATVLSIGTATPTNFVPQADYPDYYFR 39 (CHS)ITNSDHMTDLKDKFKRMCEKSMITKRHMYLTEEILKENPKMCEYM Morus alba var.APSLDARQDIVVVEVPKLGKEAAAKAIKEWGQPKSKITHLIFCTT multicaulisSGVDMPGADYQLTKLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDL Accession:AENNKGARVLVVCSEITAVTFRGPSHTHLDSLVGQALFGDGAAAV AHL83549.1ILGADPDTSVERPIFELVSAAQTILPDSEGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLVEAFTPIGISDWNSIFWIAHPGGPAILDQVEAKLGLKQEKLSATRHVLSEYGNMSSACVLFILDEVRKKSVEEGKATTGEGLEWGVLFGFGPGLTVETIVLHSLPAV Chaicone synthaseMAPPAMEEIRRAQRAEGPATVLAIGASTPPNALYQADYPDYYFRI 40 (CHS)TKSEHLTELKEKFKQMCDKSMIRKRYMYLTEEILKENPNICAFMA DendrobiumPSLDARQDIVVTEVPKLAREASARAIKEWGQPKSRITHLIFCTTS catenatumGVDMPGADYQLTRLLGLRPSVNRIMLYQQGCFAGGTVLRLAKDLA Accession:ENNAGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAAAII ALE71934.1VGSDPDLTTERPLFQLVSASQTILPESEGAIDGHLREMGLTFHLLKDVPGLISKNIQKSLVETFKPLGIHDWNSIFWIAHPGGPAILDQVEIKLGLKEEKLASSRNVLAEYGNMSSACVLFILDEMRRRSAEAGQATTGEGLEWGVLFGFGPGLTVETVVLRSVPIAGAV Chaicone isomeraseMSAITAIHVENIEFPAVITSPVTGKSYFLGGAGERGLTIEGNFIK 41 (CHI)FTAIGVYLEDVAVASLATKWKGKSSEELLETLDFYRDIISGPFEK Trifolium pratenseLIRGSKIRELSGPEYSRKVTENCVAHLKSVGTYGDAEVEAMEKFV Accession:EAFKPINFPPGASVFYRQSPDGILGVSISIHFFP PNX83855.1 Chaicone isomeraseMAAASLTAVQVENLEFPAVVTSPATGKTYFLGGAGVRGLTIEGNF 42 (CHI)IKFTGIGVYLEDQAVASLATKWKGKSSEELVESLDFFRDIISGPF Abrus precatoriusEKLIRGSKIRQLSGPEYSKKVMENCVAHMKSVGTYGDAEAAGIEE Accession:FAQAFKPVNFPPGASVFYRQSPDGVLGLSFSQDATIPEEEAAVIK XP_027366189.1NKPVSAAVLETMIGEHAVSPDLKRSLAARLPAVLSHGVFKIGN Chaicone isomeraseMAAEPSITAIQFENLVFPAVVTPPGSSKSYFLAGAGERGLTIDGK 43 (CHI)FIKFTGIGVYLEDKAVPSLAGKWKDKSSQQLLQTLHFYRDIISGP Arachis duranensisFEKLIRGSKILALSGVEYSRKVMENCVAHMKSVGTYGDAEAEAIQ Accession:QFAEAFKNVNFKPGASVFYRQSPLGHLGLSFSQDGNIPEKEAAVI XP_015942246.1ENKPLSSAVLETMIGEHAVSPDLKCSLAARLPAVLQQGIIVTPPQ HN Chaicone isomeraseMGPSPSVTELQVENVTFPPSVKPPGSTKTLFLGGAGERGLEIQGK 44 (CHI)FIKFTAIGVYLEGDAVASLAVKWKGKSKEELTDSVEFFRDIVTGP CephalotusFEKFTQVTTILPLTGQQYSEKVSENCVAFWKSVGIYTDAEAKAIE follicularisKFIEVFKEETFPPGSSILFTQSPNGALTIAFSKDGVIPEVGKAVI Accession:ENKLLAEGLLESIIGKHGVSPVAKQCLATRLSELL GAV77263.1 Flavanone 3-MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATVRDPANMKKV 45 hydroxylase (F3H)KHLLELPNAKTNLSLWKADLAEEGSFDEAIKGCTGVFHVATPMDF Abrus precatoriusESKDPENEVIKPTINGLIDIMKACMKAKTVRRLVFTSSAGTVDVT Accession:EHPKPLFDESCWSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKE XP_027329642.1NNIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAIIKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATIHDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDLGFKFKYSLEDMFTGA VETCKEKRLLSETAEISGTTQKFlavanone 3- MKDSVASATASAPGTVCVTGAAGFIGSWLVMRLLERGYIVRATVR 46hydroxylase (F3H) DPANLKKVKHLLDLPKADTNLTLWKADLNEEGSFDEAIEGCSGVFCamellia sinensis HVATPMDFESKDPENEVIKPTINGVLSIIRSCTKAKTVKRLVFTSAccession: SAGTVNVQEHQQPVFDENNWSDLHFINKKKMTGWMYFVSKTLAEK AAT66505.1AAWEAAKENNIDFISIIPTLVGGPFIMPTFPPSLITALSPITRNEGHYSIIKQGQFVHLDDLCESHIFLYERPQAEGRYICSSHDATIHDLAKLMREKWPEYNVPTEFKGIDKDLPVVSFSSKKLIGMGFEFKYSLEDMFRGAIDTCREKGLLPHSFAENPVNGNKV Flavanone 3-MVDMKDDDSPATVCVTGAAGFIGSWLIMRLLQQGYIVRATVRDPA 47 hydroxylase (F3H)NMKKVKHLQELEKADKNLTLWKADLTEEGSFDEAIKGCSGVFHVA Nyssa sinensisTPMDFESKDPENEVIKPTINGVLSIVRSCVKAKTVKRLVFTSSAG Accession:TVNLQEHQQLVYDENNWSDLDLIYAKKMTGWMYFVSKILAEKAAW KAA8531902.1EATKENNIDFISIIPTLVVGPFITPTFPPSLITALSLITGNEAHYSIIKQGQFVHLDDLCEAHIFLYEQPKAEGRYICSSHDATIYDLAKMIREKWPEYNVPTELKGIEKDLQTVSFSSKKLIGMGFEFKYSLEDMYKGAIDTCREKGLLPYSTHETPANANANANANVKKNQNENTEI Flavanone 3-MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATVRDPANKKKV 48 hydroxylase (F3H)KHLLDLPKAATHLTLWKADLAEEGSFDEAIKGCTGVFHVATPMDF Rosa chinensisESKDPENEVIKPTINGVLDIMKACLKAKTVRRLVFTASAGSVNVE Accession:ETQKPVYDESNWSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKE XP_024167119.1NNIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSIIKQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIHEIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLETGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQEVDESSVVGVKVTG Flavonoid 3′MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIGNLPHM 49 hydroxylase (F3′H)GQMPHHSLAAMARTYGPLMHLRLGFVDVIVAASASVASQLLKTHD CephalotusANFSSRPHNSGAKYIAYNYQDLVFAPYGPRWRMLRKISSVHLFSG follicularisKALDDYRHVRQEEVAVLIRALARAESKQAVNLGQLLNVCTANALG Accession:RVMLGRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIGDFIPA GAV84063.1LDWLDLQGVAAKMKNLHKRFDTFLTGLLEEHKKMLVGDGGSEKHKDLLSTLISLKDSADDEGLKLTDTEIKALLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQVLKELDTVVGRDRLVTDLDLPQLTYLQAVIKETFRLHPSTPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIARDPKEWAEPLEFRPERFLPGGEKPNVDIKGNDFEVIPFGAGRRICAGMSLGLRMVQLLTATLVHAFDWDLTSGLMPEDLSMEEAYGLTLQRA EPLMVHPRPRLSPNVYFlavonoid 3′ MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPIIGNLPH 50hydroxylase (F3′H) MGPVPHHSLAALAKVYGPLMHLRLGFVDVVVAASASVAAQFLKVHTheobroma cacao DANFSSRPPNSGAKYVAYNYQDLVFAPYGPRWRMLRKISSVHLFS Accession:GKALDDFRHVRQDEVGVLVRALADAKTKVNLGQLLNVCTVNALGR EOY22049.1VMLGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNIGDFIPALEWLDLQGVQAKMKKLHKRFDRFLSAILEEHKIKARDGSGQHKDLLSTFISLEDADGEGGKLTDTEIKALLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQVRKELDSVVGRDRLVSDLDLPNLTYFQAVIKETFRLHPSTPLSLPRMASESCEINGYHIPKGATLLVNVWAIARDPDEWKDPLEFRPERFLPGGERPNADVRGNDFEVIPFGAGRRICAGMSLGLRMVQLLAATLVHAFDWELADGLMPEKLNMEEAFGLTLQRAAPLM VHPRPRLSPRAY Flavonoid 3′MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPWPVVGNLP 51 hydroxylase (F3′H)HLGTIPHHSLAAMAKKYGPLMHLRLGFVDVVVAASASVAAQFLKT Gerbera hybridaHDANFADRPPNSGAKHIAYNYQDLVFAPYGPRWRMLRKICSVHLF Accession:STKALDDFRHVRQEEVAILARALVGAGKSPVKLGQLLNVCTTNAL ABA64468.1ARVMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIGDFIPVLDWLDLQGVTKKMKKLHAKFDSFLNTILEEHKTGAGDGVASGKVDLLSTLISLKDDADGEGGKLSDIEIKALLLNLFTAGTDTSSSTIEWAIAELIRNPQLLNQARKEMDTIVGQDRLVTESDLGQLTFLQAIIKETFRLHPSTPLSLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIWADPLEFQPTRFLPGGEKPNTDIKGNDFEVIPFGAGRRICVGMSLGLRMVQLLTATLIHAFDWELADGLNPKKLNMEEAYGLTLQRAAPLV VHPRPRLAPHVYETTKVFlavonoid 3′ MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGPRGWPVLG 52hydroxylase (F3′H) NLPQLGPKPHHTLHALSRAHGPLFRLRLGSVDVVVAASAAVAAQFPhoenix dactylifera LRAHDANFSNRPPNSGAEHIAYNYQDLVFAPYGPGWRARRKLLNVAccession: HLFSGKALEDLRPVREGELALLVRALRDRAGANELVDLGRAANKC XP_008791304.2ATNALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAGVFNVGDFVPGIGWLDLQGVVRRMKELHRRYDGFLDGLIAAHRRAAEGGGGGGKDLLSVLLGLKDEDLDFDGEGAKLTDTDIKALLLNLFTAGTDTTSSTVEWALSELVKHPDILRKAQLELDSVVGGDRLVSESDLPNLPFMQAIIKETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNVWAIARDPAVWRDPLEFRPARFLPDGGCEGMDVKGNDFGIIPFGAGRRICAGMSLGIRMVQFMTATLAHAFHWDLPEGQMPEKLDMEEAYGLT LQRATPLMVHPVPRLAPTAYQSCytochrome P450 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFF 53reductase (CPR) LLKRSSDRSKESKPVVISKPLLVEEEEEEDEVEAGSGKTKVTMFYCamellia sinensis GTQTGTAEGFAKSLAKEIKARYEKAIVKVVDLDDYAADDDQYEQKAccession: LKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY XP_028084858GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAAAIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREGSNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFAKSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDNAARFYKWFTEENERGAWLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMANGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDISGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLELLFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRLQPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMARDVHRMLHTIVEQQENVDSRKAE VIVKKLQMEGRYLRDVWCytochrome P450 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFF 54reductase (CPR) LLKRSSDRSKESKPVVISKPLLVEEEEEEDEVEAGSGKTKVTMFY CephalotusGTQTGTAEGFAKSLAKEIKARYEKAIVKVVDLDDYAADDDQYEQK follicularisLKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY Accession:GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIE GAV59576.1DDFAAWRETLWPELDQLLRDEDDANTVSTPYAAAIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREGSNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFAKSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDNAARFYKWFTEENERGAWLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMANGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDISGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLELLFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRLQPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMARDVHRMLHTIVEQQENVDSRKAE VIVKKLQMEGRYLRDVWCytochrome P450 MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAELSSMLIE 55reductase (CPR) NRQFAMIISTSIAVLIGCIVMLLWRRSGGSGSSKRAETLKPLVLKBrassica napus PPREDEVDDGRKKVTIFFGTQTGTAEGFAKALGEEARARYEKTRF Accession:KIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFY XP_013706600.1KWFTEGDDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVHVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVTPYTAAVLEYRVSIHNSADALNEKNLANGNGHAVFDAQHPYRANVAVRRELHTPESDRSCTHLEFDIAGSGLTYETGDHVGVLSDNLNETVEEALRLLDMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALTRYACLLSSPKKSALLALAAHASDPTEAERLKHLASPAGKDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAAVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKSAVPYEKSENCCSAPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFGCRNRRMDFIYEEELQRFLESGALSELSVAFSREFGPTKEYVQHKMMDKASDIWNMISQGAYVYVCGDAKGMARDVHRSLHTIAQEQGSMDSTKAESFVKNLQMSGRYLRDVW Flavonoid 3′, 5′-MALDTFLLRELAAAAVLFLISHYLIHSLLKKSTPPLPPGPKGWPF 56 hydroxylaseVGALPLLGTMPHVALAQMAKKYGPVMYLKMGTCGMVVASTPDAAR (F3′5′H)AFLKTLDLNFSNRPPNAGATHLAYNAQDMVFADYGPRWKLLRKLS CephalotusNLHMLGGKALEDWTQVRTVELGHMIQAMCEASRAKEPVVVPEMLT follicularisYAMANMIGKVILGHRVFVTQGSESNEFKDMVVELMTSAGYFNIGD Accession:FIPSIAWMDLQGIERGMKKLHKRFDALLTKMFEEHMATAHERKGN GAV62131PDLLDIVMANRDNSEGERLTTTNIKALLLNLFSAGTDTSSSIIEWSLAEMLKNPSILKRAHEEMDQVIGRNRRLEESDIKKLPYLQAICKESFRKHPSTPLNLPRVSSQACQVNGYYIPKDTRLSVNIWAIGRDPEVWENPLDFTPERFLSGKNAKIDPRGNDFELIPFGAGRRICAGTRMGIVLVEYILGTLVHSFDWSLPHGVKLNMDEAFGLALQKAVPLAA IVSPRLAPTAYVVFlavonoid 3′, 5′- MSIFLITSLLLCLSLHLLLRRRHISRLPLPPGPPNLPIIGALPFI 57hydroxylase GPMPHSGLALLARRYGPIMFLKMGIRRVVVASSSTAARTFLKTFD (F3′5′H)SHFSDRPSGVISKEISYNGQNMVFADYGPKWKLLRKVSSLHLLGS DendrobiumKAMSRWAGVRRDEALSMIQFLKKHSDSEKPVLLPNLLVCAMANVI moniliformeGRIAMSKRVFHEDGEEAKEFKEMIKELLVGQGASNMEDLVPAIGW Accession:LDPMGVRKKMLGLNRRFDRMVSKLLVEHAETAGERQGNPDLLDLV AEB96145VASEVKGEDGEGLCEDNIKGFISDLFVAGTDTSAIVIEWAMAEMLKNPSILRRAQEETDRVIGRHRLLDESDIPNLPYLQAICKEALRKHPPTPLSIPHYASEPCEVEGYHIPGETWLLVNIWAIGRDPDVWENPLVFDPERFLQGEMARIDPMGNDFELIPFGAGRRICAGKLAGMVMVQYYLGTLVHAFDWSLPEGVGELDMEEGPGLVLPKAVPLAVMATPR LPAAAYGLLDihydroflavonol 4- MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVRDPDNEKKV 58reductase (DFR) KHLVELPKAKTHLTLWKADLSDEGSFDEAIHGCTGVFHVATPMDFAcer palmatum ESKDPENEVIKPTINGVLGIMKACKKAKTVKRLVFTSSAGTVDVE Accession:EHKKPVYDENSWSDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEE AWN08247.1NSIDFISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAIIKQGNYVHLDDLCMGHIFLYEHAESKGRYFCSSHSATILELSKFLRERYPEYDLPTEYKGVDDSLENVVFCSKKILDLGFQFKYSLEDMFTGAVETCREKGLIPLTNIDKKHVAAKGLIPNNSDEIHVAAAEKTTATA Dihydroflavonol 4-MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATVRDPANMKKV 59 reductase (DFR)KHLLELPNAKTNLSLWKADLAEEGSFDEAIKGCTGVFHVATPMDF Abrus precatoriusESKDPENEVIKPTINGLIDIMKACMKAKTVRRLVFTSSAGTVDVT Accession:EHPKPLFDESCWSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKE XP_027329642.1NNIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAIIKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATIHDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDLGFKFKYSLEDMFTGA VETCKEKRLLSETAEISGTTQKDihydroflavonol 4- MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRATVRDPTNLKK 60reductase (DFR) VKPLLDLPRSNELLSIWKADLDGIEGSFDEVIRGSIGVFHVATPM DendrobiumNFQSKDPENEVIQPAINGLLGILRSCKNAGSVQRVIFTSSAGTVN moniliformeVEEHQAAAYDETCWSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFV Accession:KDNHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHYSIL AEB96144.1KQIQFVHLDDLCDAHIFLFEHPKANGRYICSSYDSTIYGLAEMLKNRYPTYAIPHKFKEIDPDIKCVSFSSKKLMELGFKYKYTMEEMFDDAIKTCREKKLIPLNTEEIVLAAEKFEEVKEQIAVK Dihydroflavonol 4-MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATVRDPANKKKV 61 reductase (DFR)KHLLDLPKAATHLTLWKADLAEEGSFDEAIKGCTGVFHVATPMDF Rosa chinensisESKDPENEVIKPTINGVLDIMKACLKAKTVRRLVFTASAGSVNVE Accession:ETQKPVYDESNWSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKE XP_024167119.1NNIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSIIKQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIHEIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLETGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQEVDESSVVGVKVTG LeucoanthocyanidinMTVSSPCVGEGQGRVLIIGASGFIGEFIAQASLDSGRTTFLLVRS 62 reductase (LAR)LDKGAIPSKSKTINSLHDKGAILIHGVIEDQEFVEGILKDHKIDI Camellia sinensisVISAVGGANILNQLTIVKAIKAVGTIKRFLPSEFGHDVDRANPVE Accession:PGLAMYKEKRMVRRLIEESGVPYTYICCNSIASWPYYDNTHPSEV XP_028127206.1IPPLDRFQIYGDGTVKAYFVDGSDIGKFTMKVVDDIRTLNKSVHFRPSCNFLNMNELSSLWEKKIGYMLPRLTVTEDDLLAAAAENIIPQSIVASFTHDIFIKGCQVNFSIDGPNEVEVSNLYPDETFRTMDECF DDFVMKMDRWNLeucoanthocyanidin MTRSPSPNGQAEKGSRILIIGATGFIGHFIAQASLASGKSTYILS 63reductase (LAR) RAAARCPSKARAIKALEDQGAISIHGSVNDQEFMEKTLKEHEIDICoffea arabica VISAVGGGNLLEQVILIRAMKAVGTIKRFLPSEFGHDVDRAEPVE Accession:PGLTMYNEKRRVRRLIEESGVPYTYICCNSIASWPYYDNTHPSEV XP_027097479.1SPPLDQFQIYGDGSVKAYFVAGADIGKFTVKATEDVRTLNKIVHFRPSCNFLNINELATLWEKKIGRTLPRVVVSEDDLLAAAEENIIPQSVVASFTHDIFIKGCQVNFPVDGPNEIEVSSLYPDEPFQTMDECFNEFAGKIEEDKKHVVGTKGKNIAHRLVDVLTAPKLCA LeucoanthocyanidinMKSTNMNGSSPNVSEETGRTLVVGSGGFMGRFVTEASLDSGRPTY 64 reductase (LAR)ILARSSSNSPSKASTIKFLQDRGATVIYGSITDKEFMEKVLKEHK Theobroma cacaoIEVVISAVGGGSILDQFNLIEAIRNVDTVKRFLPSEFGHDTDRAD Accession:PVEPGLTMYEQKRQIRRQIEKSGIPYTYICCNSIAAWPYHDNTHP ADD51357.1ADVLPPLDRFKIYGDGTVKAYFVAGTDIGKFTIMSIEDDRTLNKTVHFQPPSNLLNINEMASLWEEKIGRTLPRVTITEEDLLQMAKEMRIPQSVVAALTHDIFINGCQINFSLDKPTDVEVCSLYPDTPFRTINECFEDFAKKIIDNAKAVSKPAASNNAIFVPTAKPGALPITAICT LeucoanthocyanidinMTVSPSIASAAKSGRVLIIGATGFIGKFVAEASLDSGLPTYVLVR 65 reductase (LAR)PGPSRPSKSDTIKSLKDRGAIILHGVMSDKPLMEKLLKEHEIEIV Fragaria xISAVGGATILDQITLVEAITSVGTVKRFLPSEFGHDVDRADPVEP ananassaGLTMYLEKRKVRRAIEKSGVPYTYICCNSIASWPYYDNKHPSEVV Accession:PPLDQFQIYGDGTVKAYFVDGPDIGKFTMKTVDDIRTMNKNVHFR ABH07785.2PSSNLYDINGLASLWEKKIGRTLPKVTITENDLLTMAAENRIPESIVASFTHDIFIKGCQTNFPIEGPNDVDIGTLYPEESFRTLDECFNDFLVKVGGKLETDKLAAKNKAAVGVEPMAITATCA AnthocyaninMTQNKEPVNQGKSEHDEQRVESLASSGIESIPKEYVRLNEELTSM 66 dioxygenase (ANS)GNVFEEEKKEEGSQVPTIDIKDIASEDPEVRGKAIQELKRAAMEW ChenopodiumGVMHLVNHGISDELIDRVKVAGQTFFELPVEEKEKYANDQASGNV quinoaQGYGSKLANSASGRLEWEDYYFHLSYPEDKRDLSIWPETPADYIP Accession:AVSEYSKELRYLATKILSALSLALGLEEGRLEKEVGGLEELLLQF XP_021735950.1KINYYPKCPQPELALGVEAHTDVSALTFILHNMVPGLQLFYEGKWVTAKCVPNSIIMHIGDTIEILSNGKYKSILHRGLVNKEKVRISWAVFCEPPKEKIILKPLPDLVSDEEPARYPPRTFAQHVQYKLFRKTQ GPQTTITKN AnthocyaninMASSKVMPAPARVESLASSGLASIPTEYVRPEWERDDSLGDALEE 67 dioxygenase (ANS)IKKTEEGPQIPIVDLRGFDSGDEKERLHCMEEVKEAAVEWGVMHI Iris sanguineaVNHGIAPELIERVRAAGKGFFDLPVEAKERYANNQSEGKIQGYGS Accession:KLANNASGQLEWEDYFFHLIFPSDKVDLSIWPKEPADYTEVMMEF QCI56004.1AKQLRVVVTKMLSILSLGLGFEEEKLEKKLGGMEELLMQMKINYYPKCPQPELALGVEAHTDVSSLSFILHNGVPGLQVFHGGRWVNARLVPGSLVVHVGDTLEILSNGRYKSVLHRGLVNKEKVRISWAVFCEPPKEKIVLEPLAELVDKRSPAKYPPRTFAQHIQHKLFKKAQEQLAG GVHIPEAIQN AnthocyaninMATQVASIPRVEMLASAGIQAIPTEYVRPEAERNSIGDVFEEEKK 68 dioxygenase (ANS)LEGPQIPVVDLMGLEWENEEVFKKVEEDMKKAASEWGVMHIFNHG Magnolia sprengeriISMELMDRVRIAGKAFFDLPIEEKEMYANDQASGKIAGYGSKLAN Accession:NASGQLEWEDYFFHLIFPEDKRDMSIWPKQPSDYVEATEEFAKQL AHU88620.1RGLVTKVLVLLSRGLGVEEDRLEKEFGGMEELLLQMKINYYPKCPQPDLALGVEAHTDVSALTFILHNMVPGLQVFFDDKWVTAKCIPGALVVHIGDSLEILSNGKYRSILHRGLVNKEKVRISWAIFCEPPKEKVVLQPLPELVSEAEPARFTPRTFSQHVRQKLFKKQQDALENLKSE AnthocyaninMVSSAAVVATRVERLATSGIKSIPKEYVRPQEELTNIGNVFEEEK 69 dioxygenase (ANS)KEGPEVPTIDLTEIESEDEVVRARCHETLKKAAQEWGVMNLVNHG Prosopis albaIPEELLNQLRKAGETFFSLPIEEKEKYANDQASGKIQGYGSKLAN Accession:NASGQLEWEDYFFHLVFPEDKCDLSIWPRTPSDYIEVTSEYARQL XP_028787846.1RGLATKILGALSLGLGLEKGRLEEEVGGMEELLLQMKINYYPICPQPELALGVEAHTDVSSLTFLLHNMVPGLQLFYNGQWITAKCVPNSIFMHIGDTVEILSNGRYKSILHRGLVNKEKVRISWAVFCEPPKEKIILKPLPELVTDDEPARFPPRTFAQHIQHKLFRKCQEGLSK Anthocy anidin-3-MPQFTTNEPHVAVLAFPFGTHAAPLITIIHRLAVASPNTHFSFLN 70 O-glycotransferaseTSQSNNSIFSSDVYNRQPNLKAHNVWDGVPEGYVFVGKPQESIEL (3GT)FVKAAPETFRKGVEAAVAETGRKVSCLVTDAFFWFAAEIAGELGV CephalotusPWVPFWTAGPCSLSTHVYTDLIRKTIGVGGIEGREDESLEFIPGM follicularisSQVVIRDLQEGIVFGNLESVFSDMVHRMGIVLPQAAAIFINSFEE Accession:LDLTITNDLKSKFKQFLSIGPLNLASPPPRVPDTNGCLPWLDQQK GAV66155.1VASVAYISFGTVMAPSPPELVALAEALEASKIPFIWSLGEKLKVHLPKGFLDKTRTHGIVVPWAPQSDVLENGAVGVFITHCGWNSLLESIAGGVPMICRPFFGDQRLNGRMVQDVWEIGVTATGGPFTTEGVMGDLDLILSQARGKKMKDNISVLKTLAQTAVGPEGSSAKNYEALLNL VRLSI Anthocy anidin-3-MAPQPIDDDHVVYEHHVAALAFPFSTHASPTLALVRRLAAASPNT 71 O-glycotransferaseLFSFFSTSQSNNSLFSNTITNLPRNIKVFDVADGVPDGYVFAGKP (3GT)QEDIELFMKAAPHNFTTSLDTCVAHTGKRLTCLITDAFLWFGAHL Prunus cerasiferaAHDLGVPWLPLWLSGLNSLSLHVHTDLLRHTIGTQSIAGRENELI Accession:TKNVNIPGMSKVRIKDLPEGVIFGNLDSVFSRMLHQMGQLLPRAN AKV89253.1AVLVNSFEELDITVTNDLKSKFNKLLNVGPFNLAAAASPPLPEAPTAADDVTGCLSWLDKQKAASSVVYVSFGSVARPPEKELLAMAQALEASGVPFLWSLKDSFKTPLLNELLIKASNGMVVPWAPQPRVLAHASVGAFVTHCGWNSLLETIAGGVPMICRPFFGDQRVNARLVEDVLEIGVTVEDGVFTKHGLIKYFDQVLSQQRGKKMRDNINTVKLLAQQP VEPKGSSAQNFKLLLDVISGSTKVAnthocy anidin-3- MVFQSHIGVLAFPFGTHAAPLLTVVQRLATSSPHTLFSFFNSAVS 72O-glycotransferase NSTLFNNGVLDSYDNIRVYHVWDGTPQGQAFTGSHFEAVGLFLKA (3 GT)SPGNFDKVIDEAEVETGLKISCLITDAFLWFGYDLAEKRGVPWLA ScutellariaFWTSAQCALSAHMYTHEILKAVGSNGVGETAEEELIQSLIPGLEM baicalensisAHLSDLPPEIFFDKNPNPLAITINKMVLKLPKSTAVILNSFEEID Accession:PIITTDLKSKFHHFLNIGPSILSSPTPPPPDDKTGCLAWLDSQTR A0A482AQV3PKSVVYISFGTVITPPENELAALSEALETCNYPFLWSLNDRAKKSLPTGFLDRTKELGMIVPWAPQPRVLAHRSVGVFVTHCGWNSILESICSGVPLICRPFFGDQKLNSRMVEDSWKIGVRLEGGVLSKTATVEALGRVMMSEEGEIIRENVNEMNEKAK1AVEPKGSSFKNFNKLLEI INAPQSS Anthocy anidin-3-MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPHAVFSFFST 73 O-glycotransferaseSQSNASIFHDSMHTMQCNIKSYDISDGVPEGYVFAGRPQEDIELF (3GT)TRAAPESFRQGMVMAVAETGRPVSCLVADAFIWFAADMAAEMGLA Vitis viniferaWLPFWTAGPNSLSTHVYIDEIREKIGVSGIQGREDELLNFIPGMS Accession:KVRFRDLQEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEEL P51094DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQWLKERKPTSVVYISFGTVTTPPPAEVVALSEALEASRVPFIWSLRDKARVHLPEGFLEKTRGYGMVVPWAPQAEVLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFFGDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQILSQEKGKKLRENLRALRETADRAVGPKGSSTENFITLVDLV SKPKDV Acetyl-CoAMPPPDHKAVSQFIGGNPLETAPASPVADFIRKQGGHSVITKVLIC 74 carboxylase (ACC)NNGIAAVKEIRSIRKWAYETFGDERAIEFTVMATPEDLKVNADYI Ustilago maydisRMADQYVEVPGGSNNNNYANVDLIVDVAERAGVHAVWAGWGHASE 521NPRLPESLAASKHKIIFIGPPGSAMRSLGDKISSTIVAQHADVPC Accession:MPWSGTGIKETMMSDQGFLTVSDDVYQQACIHTAEEGLEKAEKIG XP_011390921.1YPVMIKASEGGGGKGIRKCTNGEEFKQLYNAVLGEVPGSPVFVMKLAGQARHLEVQLLADQYGNAISIFGRDCSVQRRHQKIIEEAPVTIAPEDARESMEKAAVRLAKLVGYVSAGTVEWLYSPESGEFAFLELNPRLQVEHPTTEMVSGVNIPAAQLQVAMGIPLYSIRDIRTLYGMDPRGNEVIDFDFSSPESFKTQRKPQPQGHVVACRITAENPDTGFKPGMGALTELNFRSSTSTWGYFSVGTSGALHEYADSQFGHIFAYGADRSEARKQMVISLKELSIRGDFRTTVEYLIKLLETDAFESNKITTGWLDGLIQDRLTAERPPADLAVICGAAVKAHLLARECEDEYKRILNRGQVPPRDTIKTVFSIDFIYENVKYNFTATRSSVSGWVLYLNGGRTLVQLRPLTDGGLLIGLSGKSHPVYWREEVGMTRLMIDSKTCLIEQENDPTQIRSPSPGKLVRFLVDSGDHVKANQAIAEIEVMKMYLPLVAAEDGVVSFVKTAGVALSPGDIIGILSLDDPSRVQHAKPFAGQLPDFGMPVIVGNKPHQRYTALVEVLNDILDGYDQSFRMQAVIKELIETLRNPELPYGQASQILSSLGGRIPARLEDVVRNTIEMGHSKNIEFPAARLRKLTENFLRDSVDPAIRGQVQITIAPLYQLFETYAGGLKAHEGNVLASFLQKYYEVESQFTGEADVVLELRLQADGDLDKVVALQTSRNGINRKNALLLTLLDKHIKGTSLVSRTSGATMIEALRKLASLQGKSTAPIALKAREVSLDADMPSLADRSAQMQAILRGSVTSSKYGGDDEYHAPSLEVLRELSDSQYSVYDVLHSFFGHREHHVAFAALCTYVVRAYRAYEIVNFDYAVEDFDVEERAVLTWQFQLPRSASSLKERERQVSISDLSMMDNNRRARPIRELRTGAMTSCADVADIPELLPKVLKFFKSSAGASGAPINVLNVAVVDQTDFVDAEVRSQLALYTNACSKEFSAARVRRVTYLLCQPGLYPFFATFRPNEQGIWSEEKAIRNIEPALAYQLELDRVSKNFELTPVPVSSSTIHLYFARGIQNSADTRFFVRSLVRPGRVQGDMAAYLISESDRIVNDILNVIEVALGQPEYRTADASHIFMSFIYQLDVSLVDVQKAIAGFLERHGTRFFRLRITGAEIRMILNGPNGEPRPIRAFVTNETGLVVRYETYEETVADDGSVILRGIEPQGKDATLNAQSAHFPYTTKVALQSRRSRAHALQTTFVYDFIDVLGQAVRASWRKVAASKIPGDVIKSAVELVFDEQENLREVKRAPGMNNIGMVAWLVEVLTPEYPAGRKLVVIGNDVTIQAGSFGPVEDRFFAAASKLARELGVPRLYISANSGARIGLATEALDLFKVKFVGDDPAKGFEYIYLDDESLQAVQAKAPNSVMTKPVQAADGSVHNIITDIIGKPQGGLGVECLSGSGLIAGETSRAKDQIFTATIITGRSVGIGAYLARLGERVIQVEGSPLILTGYQALNKLLGREVYTSNLQLGGPQIMYKNGVSHLTAQDDLDAVRSFVNWISYVPAQRGGPLPIMPTTDSWDRAVTYQPPRGPYDPRWLINGTKAEDGTKLTGLFDEGSFVETLGGWATSVVTGRARLGGIPVGVIAVETRTLERVVPADPANPNSTEQRIMEAGQVWYPNSAYKTAQAIWDFDKEGLPLVILANWRGFSGGQQDMYDEILKQGSKIVDGLSSYKQPVFVHIPPMGELRGGSWVVVDSAINDNGMIEMSADVNSARGGVLEASGLVEIKYRADKQRATMERLDSVYAKLSKEAAEATDFTAQTTARKALAEREKQLAPIFTAIATEYADAHDRAGRMLATGVLRSALPWENARRYFYWRLRRRLTEVAAERTVGEANPTLKHVERLAVLRQFVGAAASDDDKAVAEHLEASADQLLAASKQL KAQYILAQISTLDPELRAQLAASLKAcetyl-CoA MVDHKSLPGHFLGGNSVDTAPQDPVCEFVKSHQGHTVISKVLIAN 75carboxylase (ACC) NGMAAMKEIRSVRKWAYETFGNERAIEFTVMATPEDLKANAEYIRHesseltinella MADNYIEVPGGTNNNNYANVELIVDVAERTGVHAVWAGWGHASEN vesiculosaPRLPEMLAKSKNKCVFIGPPASAMRSLGDKISSTIVAQSADVPTM Accession:GWSGDGVSETTTDHNGHVLVNDDVYNSACVKTAEAGLASAEKIGF ORX57605.1PVMIKASEGGGGKGIRKVEDPSTFKQAFAQVQGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAKPDIFEQMEKAAVRLGKLVGYVSAGTVEYLYSHHDEKFYFLELNPRLQVEHPTTEMVSGVNLPAAQLQIAMGIPMHRIRDIRVLYGVQPNSASEIDFDLEHPTALQSQRRPMPKGHVIAVRITAENPDAGFKPSGGVMQELNFRSSTNVWGYFSVVSSGAMHEYADSQFGHIFAYGENRQQARKNMVIALKELSIRGDFRTTVEYIIRLLETPDFTDNTINTGWLDMLISKKLTAERPDTMLAVFCGAVTKAHLASVECWQQYKNSLERGQIPSKESLKTVFTVDFIYENIRYNFTVTRSAPGIYTLYLNGTKTQVGVRDLSDGGLLISLNGRSHTTYNREEVQATRLMIDGKTCLLEKESDPTQLRSPSPGKLVSLLLENGDHIRTGQAYAEIEVMKMYMPLVASEDGHVQFIKQVGATLEAGDIIGILSLDDPSRVKHALPFTGQVPKYGLPHLTGDKPHQRFTHLKQTLEYVLQGYDNQGLIQTIVKELSEVLNNPELPYSELSASMSVLSGRIPGRLEQQLHDLINQAHAQNKGFPAVDIQQAIDTFARDHLTTQAEVNAYKTAVAPIMTIAASYSNGLKQHEHSVYVDLMEQYYNVEVLFNSNQSRDEEVILALRDQHKDDLEKVINIILSHAKVNIKNNLILMLLDIIYPATSSEALDRCFLPILKHLSEIDSRGTQKVTLKAREYLILCQLPSLEERQSQMYNILKSSVTESVYGGGTEYRTPSYDAFKDLIDTKFNVFDVLPNFFYHPDSYVSLAALEVYCRRSYHAYKILDVAYNLEHQPYIVAWKFLLQSSAGGGFNNQRIASYSDLTFLLNKTEEEPIRTGAMVALKTLEELEAELPRIMTAFEEEPLPPMLMKQPPPDKTEERMENILNISIQGQDMEDDTLRKNMTTLIQAHSDAFRKAALRRITLVVCRDNQTPDYYTFRERNGYEEDETIRHIEPALAYQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDCRFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLEIVSHDYKNSDCNHLFINFIPTFAIEADEVETALKDFVDRHGKRLWKLRVTGAEIRFNIQSKRPDAPVIPLRFTVDNVSGYILKVDVYQEVKTDKNGWILKSVGKIPGAMHMQPLSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQAIHNLWAQACKADAAVKIPSQVIEAKELVLDDDNQLQAIDRAPGTNTVGMVAWLLTLRTPDYPRGRRVIAIANDITFKIGSFGVQEDLVFYKASEYARELGVPRVYLSANSGARIGLADELISRFHVAWKDEDQPGSGFEYLYLLPEEYDALIQQGDAQSVLVQEVQDKGERRFRITDIIGHTDGLGVENLRGSGLIAGATSRAYDDIFTITLVTCRSVGIGAYLVRLGQRTVQNEGQPIILTGAPALNKVLGREVYTSNLQLGGTQIMYKNGVSHLTAENDLEGINKIMQWLSFVPECRGAPLPMRAGADPIDREIEYLPPKGPSDPRFFLAGKQENGKWLSGFFDHGSFVETLSGWARTVVVGRARLGGIPMGVVAVETRTVENIVPADPANADSQEQVVMEAGGVWFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGGQRDMYNEVLKYGAQIVDALSNYKQPVFVYVVPNGELRGGAWVVVDSTINEDMMEMYADTQARGGVLEPEGIVEIKYRRPQLLATMERLDPVYSDLKRRLAALDDSQKEQADELIAQVEAREQALLPVYQQVAIQFADLHDRSGRMEAKGVIRKTLEWRTARHYFYWRVRRRLLEEYAIRKMDESRDQAKTLLQQWFQADTNLDDFDKNDQAVVAWFDAKNLLLDQRIAKLKSEKLKDHVVQLASVDQDAVVEGFSKLMESLSVDQRKEV LHKLATRF Acetyl-CoAMASTTPHDSRVVSVSSGKKLYIEVDDGAGKDAPAIVFMHGLGSST 76 carboxylase (ACC)SFWEAPFSRSNLSSRFRLIRYDFDGHGLSPVSLLDAADDGAMIPL RhodotorulaVDLVEDLAAVMEWTGVDKVAGIVGHSMSGLVASTFAAKYPQKVEK toruloidesLVLLGAMRSLNPTVQTNMLKRADTVLESGLSAIVAQVVSAALSDK NBRC10032SKQDSPLAPAMVRTLVLGTDPLGYAAACRALAGAKDPDYSTIKAK Accession:TLVVSGESDYLSNKETTEALVNDIPGAKEVQMDGVGHWHAVEDPA GEM08739.1GLAKILDGFFLQGKFSGEAKAVNGSHAVDETPKKPKYDHGRVVKYLGGNSLESAPPSNVADWVRERGGHTVITKILIANNGIAAVKEIRSVRKWAYETFGSERAIEFTVMATPEDLKVNADYIRMADQYVEVPGGTNNNNYANVDVIVDVAERAGVHAVWAGWGHASENPRLPESLAASKHKIVFIGPPGSAMRSLGDKISSTIVAQHAEVPCMDWSGQGVDQVTQSLEGYVTVADDVYQQACVHDADEGLARASRIGYPVMIKASEGGGGKGIRKVEREQDFKQAFQAVLTEVPGSPVFIMKLAGAARHLEVQVLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAKPDTFEQMEKSAVRLAKLVGYVSAGTVEFLYSAADDKFAFLELNPRLQVEHPTTEMVSGVNLPAAQLQVAMGVPLHRIRDIRTLYGKAPNGSSEIDFEFENPESAKTQRKPSPKGHVVAVRITAENPDAGFKPSMGTLQELNFRSSTNVWGYFSVGSAGGLHEFADSQFGHIFAYGSDRSESRKNMVVALKELSIRGDFRTTVEYLIKLLETDAFEQNTITTAWLDSLISARLTAERPDTTLAIICGAVTKAHLASEANIAEYKRILEKGQSPPKELLATVVPLEFVLEDVKYRATASRSSPSSWSIYVNGSNVSVGIRPLADGGLLILLDGRSYTCYAKEEVGALRLSIDSRTVLVAQENDPTQLRSPSPGKLVRYFIESGEHISKGEAYAEIEVMKMIMPLIAAEDGIAQFIKQPGATLEAGDILGILSLDDPSRVHHAKPFDGQLPALGLPSIIGTKPHQRFAYLKDVLSNILMGYDNQAIMQSSIKELISVLRNPELPYGEANAVLSTLSGRIPAKLEQTLRQYIDSAHESGAEFPSAKCRKAIDTTLEQLRPAEAQTVRNFLVAFDDIVYRYRSGLKHHEWSTLAGIFAAYAETEKPFSGKDSDVVLELRDAHRDSLDSVVKIVLSHYKAASKNSLVLALLDVVKDSDSVPLIEQVVSPALKDLADLDSKATTKVALKAREVLIHIQLPSLDERLGQLEQILKASVTPTVYGEPGHDRTPRGEVLKDVIDSRFTVFDVLPSFFQHQDQWVSLAALDTYVRRAYRSYNLLNIEHIEADAAEDEPATVAWSFRMRKAASESEPPTPTTGLTSQRTASYSDLTFLLNNAQSEPIRYGAMFSVRSLDGFRQELGTVLRHFPDSNKGKLQQQPAASSSQEQWNVINVALTVPASAQVDEDALRADFAAHVNAMSAEIDARGMRRLTLLICREGQYPSYYTVRKQDGTWKELETIRDIEPALAFQLELGRLSNFHLEPCPVENRQVHIYYATAKGNSSDCRFFVRALVRPGRLRGNMKTADYLVSEADRLVTDVLDSLEVASSQRRAADGNHISLNFLYSLRLDFDEVQAALAGFIDRHGKRFWRLRVTGAEIRIVLEDAQGNIQPIRAIIENVSGFVVKYEAYREVTTDKGQVILKSIGPQGALHLQPVNFPYPTKEWLQPKRYKAHVVGTTYVYDFPDLFRQAIRKQWKAVGKTAPAELLVAKELVLDEFGKPQEVARPPGTNNIGMVGWIYTIFTPEYPSGRRVVVIANDITFKIGSFGPEEDRYFYAVTQLARQLGLPRVYLSANSGARLGIAEELVDLFSVAWADSSRPEKGFKYLYLTAEKLGELKNKGEKSVITKRIEDEGETRYQITDIIGLQEGLGVESLKGSGLIAGETSRAYDDIFTITLVTARSVGIGAYLVRLGQRAVQVEGQPIILTGAGALNKVLGREVYSSNLQLGGTQIMYKNGVSHLTAANDLEGVLSIVQWLAFVPEHRGAPLPVLPSPVDPWDRSIDYTPIKGAYDPRWFLAGKTDEADGRWLSGFFDKGSFQETLSGWAQTVVVGRARLGGIPMGAIAVETRTIERIIPADPANPLSNEQKIMEAGQVWYPNSSFKTGQAIFDFNREGLPLIIFANWRGFSGGQQDMFDEVLKRGSLIVDGLSAYKQPVFVYIVPNGELRGGAWVVLDPSINAEGMMEMYVDETARAGVLEPEGIVEIKLRKDKLLALMDRLDPTYHALRVKSTDASLSPTDAAQAKTELAAREKQLMPIYQQVALQFADSHDKAGRILSKGCAREALEWSNARRYFYARLRRRLAEEAAVKRLGEADPTLSRDERLAIVHDAVGQGVDLNNDLAAAAAFEQGAAAITERVKLARATTVASTLAQLAQDDKEAFAASLQQVLGDKLTAADLARILA Malonyl-CoAMNANLFSRLFDGLVEADKLAIETLEGERISYGDLVARSGRMANVL 77 synthase (matB)VARGVKPGDRVAAQAEKSVAALVLYLATVRAGAVYLPLNTAYTLH RhodopseudomonasELDYFIGDAEPKLVVCDPAKREGIAALAQKVGAGVETLDAKGQGS palustrisLSEAAAQASVDFATVPREGDDLAAILYTSGTTGRSKGAMLSHDNL Accession:ASNSLTLVEFWRFTPDDVLIHALPIYHTHGLFVASNVTLFARASM WP_011661926.1IFLPKFDPDAIIQLMSRASVLMGVPTFYTRLLQSDGLTKEAARHMRLFISGSAPLLADTHREWASRTGHAVLERYGMTETNMNTSNPYDGARVPGAVGPALPGVSLRVVDPETGAELSPGEIGMIEVKGPNVFQGYWRMPEKTKAEFRDDGFFITGDLGKIDADGYVFIVGRGKDLVITGGFNVYPKEVESEIDAISGVVESAVIGVPHADLGEGVTAVVVRDKGASVDEAAVLGALQGQLAKFKMPKRVLFVDDLPRNTMGKVQKNVLR EAYAKLYAK Malonyl-CoAMVNHLFDAIRLSITSPESTFIELEDGKVWTYGAMFNCSARITHVL 78 synthase (matB)VKLGVSPGDRVAVQVEKSAQALMLYLGCLRAGAVYLPLNTAYTPA RhizobiumELEYFLGDATPKLVVVSPCAAEQLEPLARRVGTRLLTLGVNGDGS sp. BUS003LMDMASLEPVEFADIERKADDLAAILYTSGTTGRSKGAMLTHDNL Accession:LSNAQTLREHWRFTSADRLIHALPIFHTHGLFVATNVTLLAGGAI NKF42351.1YLLSKFDPDQIFALMTRATVMMGVPTFYTRLLQDERLNKANTRHMRLFISGSAPLLAETHRLFEEYTGHAILERYGMTETNMITSNPCDGARVPGTVGYALPGVSVRITDPVSGEPLAAGEPGMIEVKGPNVFQGYWNMPDKTKEEFRSDGYFTTGDIGVMETDGRISIVGRGKDLIISGGYNIYPKEIENEIDAIEGVVESAVIGVPHPDLGEGVTAIVVGQPKAHLDLTTITNNLQGRLARFKQPKNVIFVDELPRNTMGKVQKNVLR DRYRDLYLK Malonyl-CoAMANHLFDLVRANATDLTKTFIETETGLKLTYDDLMTGTARYANVL 79 synthase (matB)VGLGVKPGDRVAVQVEKSAGAIFLYLACVRAGAVFLPLNTAYTLT Ochrobactrum sp.EIEYFLGDAEPALVVCDPARRDGITEVAKKTGVPAVETLGKGQDG 3-3SLFDKAAAAPETFADVARGPGDLAAILYTSGTTGRSKGAMLSHDN Accession:LASNALTLKDYWRFGADDVLLHALPIFHTHGLFVATNTILVAGAS WP_114216069.1MLFLPKFDADKVFELMPRATTMMGVPTFYVRLVQDARLTREATKHMRLFISGSAPLLAETHKLFREKTGVSILERYGMTETNMNTSNPYDGDRVAGTVGFPLPGVALRVADPETGAAIPQGEIGVIEVKGPNVFSGYWRMPEKTAAEFRQDGFFITGDLGKIDDQGYVHIVGRGKDLVISGGYNVYPKEVETEIDGMAGVVESAVIGVPHPDFGEGVTAVVVAEKGASLDEATIIKTLEQRLARYKLPKRVIVVDDLPRNTMGKVQKNLL RDAYKGLYGG MalonateMSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGTLVADLDA 80 transporter (matC)DGIFAGFPGDLFVVLVGVTYLFAIARANGTTDWLVHAAVRLVRGR RhizobialesVALIPWVMFALTGALTAIGAVSPAAVAIVAPVALSFATRYSISPL bacteriumLMGTMVVHGAQAGGFSPISIYGSIVNGIVEREKLPGSEIGLFLAS Accession:LVANLLIAAVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGSG MBN8942514.1SDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGGAEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLTAVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTYVGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIVSAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAVSATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVYGGIVVAA VPALAWLVLVVPGFG MalonateMGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMIIGMKT 81 transporter (matC)NEIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWLVECAVRLVRGR RhizobiumIGLIPWVMFLVAAIITGFGALGPAAVAILAPVALSFAVQYRIHPV leguminosarumMMGLMVIHGAQAGGFSPISIYGGITNQIVAKAGLPFAPTSLFLSS Accession:FFFNLAIAVLVFFVFGGARVMKHDPASLGPLPELHPEGVSASIRG AAC83457.1HGGTPAKPIREHAYGTAADTATTLRLNNERITTLIGLTALGIGALVFKFNVGLVAMTVAVVLALLSPKTQKAAIDKVSWSTVLLIAGIITYVGVMEKAGTVDYVANGISSLGMPLLVALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAIGVVAAIAISTTIVDTSPFSTNGALVVANAPDDSREQVLRQLLIYSALIAIIGPIVAWLVFVVPGLV MalonateMNIEILSIGLLVAIFIIATIQPINMGVLAFGCTFVLGSLIIGMKP 82 transporter (matC)ADIFAGFPADLFLTLVAVTYLFAIAQINGTIDWLVERSVRMVRGR Agrobacterium vitisVGWIPWVMFLVAAIITGFGALGPAAVAILAPVALSFAVQYRIHPV Accession:LMGLMVIHGAQAGGFSPISIYGGITNQIVAKAGLPFAPTSLFLSS WP_180575084.1FFFNLAIAVLIFFIFGGLSILKQRSSVKGPLPELHPEGISASIKGHGGTPAKPFREHAYGTAADTQSKVRLTTEKVTTLIGLTALGVGALVFKFNVGLVAITVAVLLALLSPTTQKAAIDKVSWSTVLLISGIITYVGVMEKAGTIDYVAHGISSLGMPLLVALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAVGVVAAIAISTTIVDTSPFSTNGALVVANAPDDQRDKVMRQMLIYSALIALIGPVIAWLVFVVPGII MalonateMSIEILSILLLVAMFVIATIQPINMGALAFACTFVLGSLIIGMKT 83 transporter (matC)SDIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWLVECAVRMVRGH Neorhizobium sp.VAWIPWVMFVVAAITGFGALGPAAVAILAPVALSFAVQYRIHPVM Accession:MGLMVIHGAQAGGFSPISVYGGITNQIVAKAGLPFAPTSLFLSSF WP_105370917.1FFNLAIAVLVFFVFGGARIMKQAAGPTGPLPELHPEGVSAAIRGHGGTPAKPIREHAYGTAADTLQTLRLTPEKVFTLIGLTALGIGALVFKFNVGLVAITVAVALALISPKTQKAAVDKVSWSTVLLIAGIITYVGVLEKAGTVNYVANGISSLGMPLLVALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAVGVVAAIAISTTIVDTSPFSTNGALVVANAPDETREQVLRQLLIYSALIAIIGPVVAWLVFVVPGLV Malonate CoA-MTTWNQKQQRKAQKLAKACDSGFDKYVPHERIIALLETVIDRGDR 84 transferase (MdcA)VCLEGNNQKQADFLSKSLSSCNPDIVNGLHIVQSVLALPSHIDVF MoraxellaERGIASKVDFSFAGPQSLRLAQLVQAQKITIGAIHTYLELYGRYF catarrhalisIDLTPNVALITAHAADKRGNLYTGANTEDTPAIVEATTFKSGIVI Accession:AQVNEIVDELPRVDIPSDWVDYYTQSPKHNYIEPLFTRDPAQITE WPO64617969.1IQILMAMMAIKGIYAPYKINRLNHGIGFDTAAIELLLPTYAESLGLKGEICTHWALNPHPTLIPAIESGFIHSVHSFGSEVGMENYVKARSDVFFTGADGSMRSNRAFSQTAGLYACDLFIGSTLQIDLQGNSSTATADRIAGFGGAPNMGSDPHGRRHASYAYMKAGREAVDGSPIKGRKLVVQMVETYREHMQSVFVNELDAFKLQQKMGADLPPIMIYGDDVTHIVTEEGIANLLLCRTPDEREQAIRGVAGYTPIGLGRDDTMVARLRERKVIQRPEDLGINPMHATRDLLAAKSVKDLVRWSDRLYEPPS RFRNW Malonate CoA-MNAPQPRQWDSLRQNRARRLERAASLGLAGQNGKEIPVDRIIDLL 85 transferase (MdcA)EAVIQPGDRVCLEGNNQKQADFLSESLADCDPARINHLSMVQSVL DechloromonasALPSHVDLFERGLATRLDFSFSGPQGARLAKLVQEQRIEIGAIHT aromaticaYLELFGRYFMDLTPNVALIAAQAADAEGNLYLGPNTEDTPAIVEA Accession:TAFKGGIVIAQVNERLDKLPRVDVPADWVDFTVLAPKPNYIEPLF WP_011289741.1TRDPAQITEVQVLMAMMAIKGIYAEYGVTRLNHGIGFDTAAIELLLPTYAADLGLKGKICTHWALNPHPTLIPAIEAGFVESVHCFGSEVGMDDYISARSDIFFTGADGSMRSNRAFSQTAGLYACDMFIGSTLQMDLAGNSSTATLGRITGFGGAPNMGSDPHGRRHASPAWLKAGREAYGPQAIRGRKLVVQMVETFREHMAPVFVDDLDAWKLQASMGSDLPPIMIYGDDVSHIVTEEGIANLLLCRTPAEREQAIRGVAGFTPVGMARDKGTVENLRDRGIIRRPEDLGIDPRQASRDLLAARSIKDLVRC SGGLYAPPSRFRNWMalonate CoA- MSRQWDTQADSRRQRLQRAAALAPQGRVVAADDVVALLEAVIEPG 86transferase (MdcA) DRVCLEGNNQKQADFLARCLTEVDPARVHDLHMVQSVLSLAAHLDPseudomonas VFERGIAKRLDFSFSGPQAARLAGLVSEGRIEIGAIHTYLELFGR cissicolaYFIDLTPRIALVTAQAADRHGNLYTGPNTEDTPVIVEATAFKGGI Accession:VIAQVNEILDTLPRVDIPADWVDFVTQAPKPNYIEPLFTRDPAQI WP_078590875.1SEIQVLMAMMAIKGIYAEYGVDRLNHGIGFDTAAIELLLPTYAQSLGLKGKICRHWALNPHPALIPAIESGFVQSVHSFGSELGMENYIAARPDIFFTGADGSMRSNRALSQTAGLYACDMFIGSTLQIDLQGNSSTATRDRIAGFGGAPNMGSDARGRRHASAAWLKAGREAATPGEMPRGRKLVVQMVETFREHMAPAFVDRLDAWELAERANMPLPPVMIYGDDVSHVLTEEGIANLLLCRTPEEREQAIRGVSGYTAVGLGRDKRMVENLRDRGVIKRPDDLGIRPRDATRDLLAARTVKDLVRWSGGLYD PPKRFRNW Malonate CoA-MNKIYREKRSWRTRRDRKAKRIEHMKQIAKGKIIPTEKIVEALTA 87 transferase (MdcA)LIFPGDRVVIEGNNQKQASFLSKALSQVNPEKVNGLHIIMSSVSR GeobacillusPEHLDLFEKGIARKIDFSYAGPQSLRMSQMLEDGKLVIGEIHTYL subterraneusELYGRLFIDLTPSVALVAADKADASGNLYTGPNTEETPTLVEATA Accession:FRDGIVIAQVNELADELPRVDIPGSWIDFVVAADHPYELEPLFTR WP_184319829.1DPRLITEIQILMAMMVIKGIYERHNIQSLNHGIGFNTAAIELLLPTYGESLGLKGKICKHWALNPHPTLIPAIETGWVESIHCFGGEVGMEKYIAARPDIFFTGKDGNLRSNRTLSQVAGQYAVDLFIGSTLQIDRDGNSSTVTNGRLAGFGGAPNMGHDPRGRRHSSPAWLDMITSDHPAAKGRKLVVQMVETFQKGNRPVFVESLDAIEVGRSARLATTPIMIYGEDVTHIVTEEGIAYLYKASSLEERRQAIAAIAGVTPIGLERDPRKTEQLRRDGVVAFPEDLGIRRTDAKRSLLAAKSIEELVEWSEGL YEPPARFRSWPantothenate kinase MLLTIDVGNTHTVLGLFDGEEIVEHWRISTDSRRTADELAVLLQG 88(CoaX) LMGTHPLLGMELGEGIDGIAICSTVPAVLHELREVSRRYYGDVPA Streptomyces sp.ILVEPGVKTGVPILMDNPKEVGTDRIINAVAAQHLYGGPAIVVDF CLI2509GTATTFDAVSARGEYTGGVIAPGIEISVEALGLRGAQLRKIELAR Accession:PRSVIGKSTVEAMQSGILYGFAGQVDGVVQRMACELAPDPADVTV WP_095682415.1IATGGLAPMVLGEAAVIDHHEPWLTLIGLRLVYERNAGRR Pantothenate kinaseMTKLWLDLGNTRLKYWLTDDSGQVLDHAAEQHLQAPAELLKGLTF 89 (CoaX)RLERLNPDFIGVSSVLGQAVNNHVAESLERLQKPFEFAQVHAKHA StreptomycesLMSSDYNPAQLGVDRWLQMLGIIEPSKKQCVIGCGTAVTIDLVDQ cinereusGHHLGGYIFPSIYLQRESLFSGTRQISIIDGTFDSIDSGTNTQDA Accession:VHHGIMLSIVGAINETIHRYPQFEITMTGGDAHTFEPHLSASVEI WP_188874884.1RQDLVLAGLQRFFAAKNNTKNQN Pantothenate kinaseMLLTIDVGNTQTTLGLFDGEEVVDHWRISTDPRRTADELAVLMQG 90 (CoaX)LMGRQPGGAGRERVDGLAICSSVPAVLHELREVTRRYYGDLPAVL KitasatosporaVAPGVKTGVHVLMDNPKEVGADRIVNALAANHLYGGPCIVVDFGT kifunensisATTFDAINERGDYVGGAIAPGIEISVEALGVRGAQLRKIELAKPR Accession:NVIGKNTVEGMQSGVLYGFAGQVDGLVTRMAKELSPTDPEDVQVI WP_184936930.1ATGGLAPLVLDEASSIDVHEPWLTLIGLRLVYERNTAS glutamyl-tRNAMTLLALGINHKTAPVSLRERVTFSPDTLDQALDSLQALPMVQGGV 91 reductase (hemA)VLSTCNRTEIYLSVEEQDNLREALIRWLCEYHNLNEEDLRNSLYW CitrobacterHQDNDAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQKGHQNA freundiiSALERMFQKSFSVAKRVRTETDIGSSAVSVAFAACTLARQIFESL Accession:STVTVLLVGAGETIELVARHLREHKVKKMIIANRTRERAQVLADE NTY05430.1VGAEVISLSDIDARLQDADIIISSTASPLPIIGKGMVERALKNRRNQPMLLVDIAVPRDVEPEVGKLSNAYLYSVDDLQSIISHNLAQRKAAAVEAETIVEQEASEFMAWLRAQGASDTIREYRSQSEQIRDELTAKALAALQQGGDAQAIMQDLAWKLTNRLIHAPTKSLQQAARDGDS ERLNILRDSLGLEglutamyl-tRNA MTLLALGINHKTAPVSLRERVTFSPETIEQALSSLLQQPLVQGGV 92reductase (hemA) VLSTCNRTELYLSVEQQENLQEQLVKWLCDYHHLSADEVRKSLYWPseudomonas HQDNAAVSHLMRVASGLDSLVVGEPQILGQVKKAFAESQHGQAVS reactansGELERLFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESL Accession:SDVSVLLVGAGETIELVARHLREHKVRHMMIANRTRERAQVLASE NWA43040.1VGAEVITLQDIDARLADADIIISSTASPLPIIGKGMVERALKARRNQPMLMVDIAVPRDIEPEVGKLANAYLYSVDDLHSIIQNNMAQRKAAAVQAESIVEQESSNFMAWLRSQGAVEIIRDYRSRADLVRAEAEAKALAAIAQGADVSAVIHELAHKLTNRLIHAPTRSLQQAASDGDV ERLQILRDSLGLDQQglutamyl-tRNA MTLLALGINHKTAPVALREKVSFSPDTMGDALNNLLQQPAVRGGV 93reductase (hemA) VLSTCNRTELYLSMEDKENSHEQLIRWLCQYHQIEPNELQSSIYWGammaproteobacteria HQDNQAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQNYDSLSAccession: SELERLFQKSFSVAKRVRTETQIGANAVSVAFAACTLARQIFESL WP_193016510.1SSLTILLVGAGETIELVARHLREHQVKKIIIANRTKERAQRLASEVDAEVITLSEIDECLAQADIVISSTASPLPIIGKGMVERALKKRRNQPMLLVDIAVPRDIEQDVEKLNNVYLYSVDDLEAIIQHNREQRQAAAVQAEHIVQQESGQFMDWLRAQGAVGAIREYRDSAETLRAEMTEKAITLIQNGADAEKVIQQLSHQLMNRLIHTPTKSLQQAASDGDI ERLNLLRESLGITHN5-aminolevulinic MGPALDVRGKQLAAGYASVAGQADVEKIHQDQGITIPPNATVEMC 94acid synthase PHAKAARDAARIAEDLAAAAASKQQPAKKAGGCPFHAAQAQAQAK (ALAS)PAAAPKETVATADKKGKSPRAAGGFDYEKFYEEELDKKHQDKSYR SchizophyllumYFNNINRLAARFPTAHTAKVTDEVEVWCSNDYLGMGGNPVVLETM commune H4-8HRVLDKYGHGAGGTRNIAGNGALHLSLEQELARLHRKEGALVFTS Accession:CYVANDATLSTLGSKMPGCVIFSDRMNHASMIQGIRHSGTKKVIF XP_003036856.1EHNDLADLEKKLAEYPKETPKIIAFESVYSMCGSIGPIKEICDLAEKYGAITFLDEVHAVGLYGPRGAGVAEHLDYDLHKAAGDSPDAIPGTVMDRVDIITGTLGKSYGAIGGYIAGSARFVDMIRSYAPGFIFTTSLPPATVAGAQASVVYQKEYLGDRQLKQVNVREVKRRFAELDIPVVPGPSHIVPVLVGDAALAKQASDKLLAEHDIYVQAINYPTVARGEERLRITVTQRHTLEQMDHLIGAVDQVFNELNINRVQDWKRLGGRASVGVPGGQDFVEPIWTDEQVGLADGSAPLTLRNGQPNEVSHDAVVAARSRFDWLLGPIPSHIQAKRLGQSLEGTPIAPLAPKQSSGLKL PVEEMTMGQTIAVAA5-aminolevulinic MDKIARFKQTCPFLGRTKNSTLRNLSTSSSPRFPSLTALTERATK 95acid synthase CPVMGPALNVRSKEIVAGYASVAANSDVALIHKEKGVFPPPGATV (ALAS)EMCPHASAARAAARMADDLAAAAEKKKGHFTSAAPRDEAAQAAAA CrassisporiumGCPFHVKAAADAAAARKAAAAPAPVKAKEDGGFNYESFYVNELDK junariophilumKHQDKSYRYFNNINRLAAKFPVAHTSNVKDEVEVWCANDYLGMGN Accession:NPVVLETMHRTLDKYGHGAGGTRNIAGNGAMHLSLEQELATLHRK KAF8165006.1PAALVFSSCYVANDATLSTLGAKLPGCIFFSDTMNHASMIQGMRHSGAKRVLFKHNDLEDLENKLKQYPKDTPKVIAFESVYSMCGSIGPIKEICDLAEQYGALTFLDEVHAVGLYGPRGAGVAEHLDYDAHVAAGESPHPIKGSVMDRVDIITGTLGKAYGAVGGYIAGSDDFVDMIRSYAPGFIFTTSLPPATVAGARASVVYQKHYVGDRQLKQVNVREVKRRFAELDVPVVPGPSHIVPVLVGDAALAKAASDKLLAEHNIYVQSINYPTVARGEERLRITVTPRHTLEQMDKLVRAVDKIFAELKINRLADWKALGGRAGVGLTAGAEEAHVDPMWTEEQLGLLDGTSPRTLRNGEAAVVDAMAVGQARAVFDNLLGPISGKLQSERSVLASSTPAAANPARPAARKVVKMKTGGVPMSEDIPLPPPDVSASA 5-aminolevulinicMDKLSSLSRFKASCPFLGRTKTSTLRTLCTSSSPRFPSISILTER 96 acid synthaseATKCPVMGPALNVRSKEITAGYASVAGSSEVDQIHKQQGVTVPVN (ALAS)ATVEMCPHASAARAAARMADDLAAAAAQKKVGSGASSAKAAAAGC DendrothelePFHKSVAAGASASTASKPSAPIHKASVPGGFDYDNFYNNELEKKH bispora CBSKDKSYRYFNNINRLASKFPVAHTGDVKDEVQVWCSNDYLGMGNNP 962.96VVLETMHRTLDKYGHGAGGTRNIAGNGALHLGLEQELAALHRKEA Accession:ALVFSSCYVANDATLSTLGSKLPGCILFSDKMNHASMIQGMRHSG THV05492.1AKKVIFNHNDLEDLENKLKQYPKETPKIIAFESVYSMCGSIGPIKEICDLAEKYGALTFLDEVHAVGLYGPHGAGVAEHLDYNAQKAAGKSPEPIPGSVMDRVDIITGTLGKAYGAVGGYIAGSMDFVDTIRSYAPGFIFTTSLPPATVSGAQASVAYQKEYLGDRQLKQVNVREVKRRFAELDIPVIPGPSHILPVLVGDAALAKAASDKLLTDHDIYVQSINYPTVAVGEERLRITVTPRHTLEQMDKLVRAVNQVFTELNINRISDWKVAGGRAGVGMGVESVEPIWTDEQLGITDGTTPKTLRDGQRFLVDAQGVTAARGRFDTLLGPMSGSLQANPTLPLVD DELKVPLPTLVAAAA 5-aminolevulinicMDYAQFFNTALDRLHTERRYRVFADLERIAGRFPHALWHSPKGKR 97 acid synthaseDVVIWCSNDYLGMGQHPKVVGAMVETATRVGTGAGGTRNIAGTHH (ALAS)PLVQLEAELADLHGKEASLLFTSGYVSNQTGIATIAKLIPNCLIL BradyrhizobiumSDELNHNSMIEGIRQSGCERVVFRHNDLADLEEKLKAAGPNRPKL japonicumIACESLYSMDGDVAPLAKICDLAEKYGAMTYVDEVHAVGMYGPRG Accession:GGIAERDGVMHRIDILEGTLAKAFGCLGGYIAANGQIIDAVRSYA AOAOA3YXD2PGFIFTTALPPAICSAATAAIRHLKTSNWERERHQDRAARVKAILNAAGLPVMSSDTHIVPLFIGDAEKCKQASDLLLEQHGIYIQPINYPTVAKGTERLRITPSPYHDDGLIDQLAEALLQVWDRLGLPLKQKS LAAE Cytochrome b5MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLEEHPGGEEV 98 Petunia x hybrida,LIESAGKDATKEFQDIGHSKAAKNLLFKYQIGYLQGYKASDDSEL Accession:ELNLVTDSIKEPNKAKEMKAYVIKEDPKPKYLTFVEYLLPFLAAA AAD10774.1 FYLYYRYLTGALQF

TABLE 12 Glossary of abbreviations Abbreviation Full Name 3GTanthocyanidin-3-O-glycotransferase 4CL 4-coumarate-CoA ligase ACCacetyl-CoA carboxylase ACOT acyl-CoA thioesterase acpP acyl carrierprotein ACS acetyl-CoA synthase adhE aldehyde-alcohol dehydrogenase ADPadenosine diphosphate ALA 5-aminolevulinic acid ALAS ALA synthase ANSanthocyanin dioxygenase aroG DAHP synthase aroK shikimate kinase aroLshikimate kinase ATP adenosine triphosphate C3G cyanidin-3-O-glycosideC4H cinnimate-4-hydroxylase CHI chalcone isomerase CHS chalcone synthaseCoA coenzyme A CPR cytochrome P450 Reductase DAD diode array detectorDAHP deoxy-d-arabino-heptulosonate-7-phosphate DctPQM a malonatetransporter DFR dihydroflavonol 4-reductase DHK dihydrokaempferol DHMdihydromyricein DHQ dihydroquercetin DMSO dimethyl sulfoxide E4Perythrose-4-phosphate F3′H flavonoid 3′ hydroxylase F3H flavanone3-hydroxylase fabB beta-ketoacyl-ACP synthase I fabD malonyl-coA-ACPtransacylase fabF beta-ketoacyl-ACP synthase II FadA 3-ketoacyl-CoAthiolase FadB fatty acid oxidation complex subunit alpha FadE acyl-CoAdehydrogenase GltX glutamyl-tRNA synthetase hemA glutamyl-tRNA reductasehemL glutamate-1-semialdehyde aminotransferase HPLC high performanceliquid chromatography ldhA lactate dehydrogenase LAR leucoanthocyanidinreductase matB malonyl-CoA synthase matC malonate transporter mdcAmalonate coA-transferase mdcC acyl-carrier protein, subunit of mdc mdcDmalonyl-CoA decarboxylase, subunit of mdc mdcE co-decarboxylase, subunitof mdc pABA para-aminobenzoic acid PAL phenylalanine ammonia-lyase PanKpantothenase kinase Pdh pyruvate dehydrogenase PEP phosphoenolpyruvatepHBA para-hydroxybenzoic acid PHE phenylalanine pheA chorismatemutase/prephenate dehydrogenase poxB pyruvate dehydrogenase ppsAphosphoenolpyruvate synthase TAL tyrosine ammonia-lyase TCAtricarboxylic acid cycle tesA thioesterase I tesB thioesterase II tktAtransketolase TRP tryptophan TYR tyrosine TyrA chorismate mutase tyrRtranscriptional regulator ybgC a thioesterase yciA a thioesterase ydiBQUIN/shikamate dehydrogenase ackA-pta Acetate kinase-phosphateacetyltransferase

1. An engineered host cell, wherein the engineered host cell comprisesone or more genetic modifications to increase the production ofdihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/orpentahydroxyflayaone (PHF), wherein the engineered host cell comprisescytochrome P450 reductase (CPR) and at least one offlavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), orflavonoid 3′,5′-hydroxylase (F3′5′H).
 2. The cell of claim 1, whereinthe precursor for increase in production of dihydroquercetin (DHQ),dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone(PHF) is naringenin and/or dihydrokaempferol (DHK).
 3. The engineeredhost cell of claim 1, wherein the flavonoid 3′-hydroxylase (F3′H) istruncated to remove the N-terminal leader sequence.
 4. The engineeredhost cell of claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H)is truncated to remove the N-terminal leader sequence.
 5. The engineeredhost cell of claim 1, wherein the cytochrome P450 reductase (CPR) istruncated to remove the N-terminal leader sequence.
 6. The engineeredhost cell of claim 1, wherein the flavonoid 3′-hydroxylase (F3′H) isfused with cytochrome P450 reductase (CPR).
 7. The engineered host cellof claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is fusedwith cytochrome P450 reductase (CPR).
 8. The engineered host cell ofclaim 1, wherein the flavanone-3′-hydroxylase (F3′H) has an amino acidsequence at least 80% identical to the polypeptide set forth in SEQ IDNO.
 8. 9. The engineered host cell of claim 1, wherein the cytochromeP450 reductase (CPR) has an amino acid sequence at least 80% identicalto the polypeptide set forth in SEQ ID NO.
 9. 10. The engineered hostcell of claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) has anamino acid sequence at least 80% identical to the polypeptides selectedfrom a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and(iii) SEQ ID NO.
 57. 11. The engineered host cell of claim 1, whereinthe engineered host cell further comprises cytochrome b₅.
 12. Theengineered host cell of claim 11, wherein the cytochrome b₅ has an aminoacid sequence at least 80% identical to the polypeptide set forth in SEQID NO.
 98. 13. The engineered host cell of claim 1, wherein theflavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80%identical to the polypeptides selected from a group consisting of: (i)SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO.47, and (v) SEQ ID NO.
 48. 14. A method of increasing the production ofdihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/orpentahydroxyflayaone (PHF) comprising an engineered host cell, whereinthe engineered host cell comprises cytochrome P450 reductase (CPR) andat least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase(F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H).
 15. The method of claim14, wherein the precursor for increase in production of dihydroquercetin(DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/orpentahydroxyflayanone (PHF) are naringenin and/or dihydrokaempferol(DHK).
 16. The method of claim 14, wherein the flavonoid 3′-hydroxylase(F3′H) is truncated to remove the N-terminal leader sequence.
 17. Themethod of claim 14, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) istruncated to remove the N-terminal leader sequence.
 18. The method ofclaim 14, wherein the cytochrome P450 reductase (CPR) is truncated toremove the N-terminal leader sequence.
 19. The method of claim 14,wherein the flavonoid 3′-hydroxylase (F3′H) is fused with cytochromeP450 reductase (CPR).
 20. The method of claim 14, wherein the flavonoid3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase(CPR).
 21. The method of claim 14, wherein the flavanone-3′-hydroxylase(F3′H) has an amino acid sequence at least 80% identical to thepolypeptide set forth in SEQ ID NO.
 8. 22. The method of claim 14,wherein the cytochrome P450 reductase (CPR) has an amino acid sequenceat least 80% identical to the polypeptide set forth in SEQ ID NO.
 9. 23.The method of claim 14, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H)has an amino acid sequence at least 80% identical to the polypeptidesselected from the group consisting of: (i) SEQ ID NO. 10, (ii) SEQ IDNO. 56, and (iii) SEQ ID NO.
 57. 24. The method of claim 14, wherein theengineered host cell further comprises cytochrome b₅.
 25. The method ofclaim 24, wherein the cytochrome b₅ has an amino acid sequence at least80% identical to the polypeptide set forth in SEQ ID NO.
 98. 26. Themethod of claim 14, wherein the flavanone-3-hydroxylase (F3H) has anamino acid sequence at least 80% identical to the polypeptides selectedfrom a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii)SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.